CN103370334B - Modified human tumor necrosis factor receptor-I polypeptide or fragment thereof and preparation method thereof - Google Patents

Abstract

The present invention relates to modified human tumor necrosis factor receptor-1 polypeptides or fragments thereof that bind to tumor necrosis factor in vivo or ex vivo. The modified human tumor necrosis factor receptor-1 polypeptide or fragment of the present invention exhibits improved tumor necrosis factor binding and improved protease resistance.

Description

Modified human tumor necrosis factor receptor-I polypeptide or fragment thereof and preparation method thereof

technical field

The present invention relates to modified human tumor necrosis factor receptor-I polypeptides or fragments thereof capable of binding to tumor necrosis factor in vivo or in vitro and methods for their production.

Background technique

Inflammation is the body's defense response to antigenic stimuli. Inflammatory responses can be pathologically exacerbated when inflammation occurs even after removal of harmful antigenic material or when the inflammatory response is induced by inappropriate stimuli such as self-antigens. This inflammatory response involves a variety of cytokines. Specifically, tumor necrosis factor (hereinafter referred to as "TNF") was identified as a cytokine that functions to control inflammation.

TNF was originally discovered as a protein that eliminates tumor cells (Carswell et al., PNAS 72:3666-3670, 1975). TNF is a class of cytokines produced by numerous cell types, including monocytes and macrophages, and is directly involved in inflammatory responses. At least two TNFs (TNF-α and TNF-β) have been described previously, and each acts as a trimeric molecule and is thought to initiate intracellular signaling by crosslinking receptors (Engelmann et al., J. Biol. Chem., 265:14497-14504). TNF induces inflammatory responses in vivo to regulate cell-mediated immune responses and defense mechanisms and has important physiological effects on many different target cells (Selby et al., Lancep 1:483, 1988). However, excess TNF has been shown to lead to pathological conditions such as rheumatoid arthritis, degenerative arthritis, psoriasis or Crohn's disease, and inhibition of TNF has been shown to be therapeutic for these diseases (Feldmann et al., Nat. Med. 9 :1245-1250, 2003).

Tumor necrosis factor receptor (hereinafter "TNFR") is a cytokine receptor that binds to TNF.

Two types of TNFR have been identified, called p55-TNFRI and p75-TNFRII. Almost every mammalian cell can show expression of TNFRI, whereas TNFRII expression is mostly restricted to cells of the immune system and endothelial cells.

A 28% amino acid sequence similarity was shown between the two TNF receptors. Both receptors have extracellular domains and have 4 cysteine-rich domains.

The cytoplasmic portion of TNFRI contains a "death domain" that initiates apoptotic signaling. TNFRII has no death domain and its function is still not clearly defined. Furthermore, TNFRI and TNFRII show differences in affinity for TNF-α as a ligand. TNFRI is known to show 30-fold or higher affinity than TNFRII (Tartaglia et al., J. Biol. Chem. 268:18542-18548, 1993). Due to such differences in affinity, various attempts have been made to develop drugs involving TNFRIs.

TNFR attached to the cell surface is cleaved by proteases to generate soluble TNFR. Soluble TNFR neutralizes excess TNF to control TNF levels. In conditions such as autoimmune diseases and chronic inflammation, excessive levels of TNF exceed the capacity for self-regulation.

To artificially control TNF signaling, various strategies to block TNF have been attempted, including inhibition of TNF synthesis, inhibition of TNF secretion or shedding, and inhibition of TNF signaling. Among TNF blocking methods, a method of blocking TNF signaling by preventing TNFR from binding to TNF has been used in drug development. For example, etanercept (made by fusing the extracellular region of TNFRII to the Fc region of an antibody) and antibodies that bind TNF (adalimumab and infliximab) have been used in It is used globally as a therapeutic agent for rheumatoid arthritis, psoriasis, ankylosing spondylitis, etc.

Lenercept, which is a fusion protein of antibody Fc and TNFRI ectodomain produced by using the same technology as the anti-rheumatoid arthritis drug etanercept, has completed phase II clinical trials in Europe and the United States Test (Furst et al., J. Rheumatol. 30:2123-2126, 2003). In addition, TNFRI dimers and PEGylated soluble TNFRI molecules have been studied (Carl et al., Ann. Rheum. Dis. 58:173-181, 1999).

In addition, modification of the amino acid sequence has been investigated as a means of reducing the immunogenicity of TNFRI and increasing the ability of TNFRI to bind to TNF. Specifically, a TNFRI mutant that reduces the appearance of antibodies against TNFRI by partially substituting its amino acid sequence and a TNFRI mutant that has an increased ability of TNFRI to bind to TNF are known (US Pat. No. 7,144,987).

Studies have been actively conducted to find the active site responsible for the binding of TNFR to TNF, and it is known that the fourth domain of TNFR is not necessary for TNF binding, and when the second and third domains are deleted, it results in loss of TNF binding activity ( Corcoran et al., Eur. J. Biochem. 233:831-840, 1994). In addition, a certain region of the third domain used for binding of TNFRI to TNF can be deficient, and the amino acid sequence consisting of amino acid residues 59 to 143 of the human TNFRI polypeptide (SEQ ID NO: 1) is known to be a biological marker for TNFRI. area of biological activity (US Patent No. 6,989,147).

Therefore, since the binding of TNFRI to TNF occurs in this region, other regions may contain a large number of added groups, eliminated groups or substituted groups. Meanwhile, in order to enhance bioavailability, TFNRI is used in the form of TNFRI polypeptide fragments instead of full-length TNFRI. For the purpose of producing effective injectable and oral formulations that minimize protease cleavage and enhance cell permeability, it is desirable to make TFNRI as small as possible.

Since protein therapeutics are cleared through general processes such as metabolism during circulation in vivo, glomerular filtration, and protease action in the gastrointestinal tract, tissues, and blood, it is difficult to simultaneously deliver protein therapeutics to target sites in vivo. Preserves the intrinsic activity of the protein. Specifically, drug clearance by proteases has a significant impact on the half-life of protein therapeutics when administered orally, intravascularly or intramuscularly, etc.

Human tumor necrosis factor inhibitors in the form of injections have been developed, which are one of protein therapeutics and control TNF in vivo, but administration of injections has problems associated with pain and risk of infection. Therefore, another approach, such as reducing the frequency of injections or oral administration, is needed. Enhancing the stability of human tumor necrosis factor inhibitors is crucial for this purpose, but protease degradation constitutes a formidable obstacle for this purpose.

Meanwhile, although wild-type TFNRI regulates the intracellular effect of TNF-α by binding to TNF-α, the binding ability of TNFRI is not as high as that of antibodies. Therefore, wild-type TNFRI is less effective at inhibiting TNF-α than the antibody. The development of protein therapeutics using TNFRIs requires the selection of TNFRIs capable of strong coupling to TNF-α.

Therefore, one of the main goals in developing protein therapeutics is to improve biological activity and protease resistance.

This theme was implemented as part of the Industrial Original Technology Development Program (Subject ID No. 10040233), funded by the Korean Government Ministry of Knowledge Economy, Korea Industrial Technology Evaluation Institute.

Contents of the invention

technical problem

It is an object of the present invention to provide modified human tumor necrosis factor receptor-I (TNFRI) polypeptides or fragments thereof which have increased TNF binding capacity and improved proteases present in the gastrointestinal tract, cytoplasm and blood, in vivo or ex vivo resistance.

Technical solutions

Unless otherwise stated, all technical and scientific terms used in this specification, examples and appended claims have the meanings defined below.

As used herein, the term "human tumor necrosis factor receptor-I" or "human tumor necrosis factor receptor-I polypeptide" (hereinafter referred to as "TNFRI" or "TNFRI polypeptide") refers to a human-derived 455-amino acid And a polypeptide capable of binding to TNF.

As used herein, the term "human tumor necrosis factor receptor-1 fragment" or "human tumor necrosis factor receptor-1 polypeptide fragment" (hereinafter referred to as "TNFRI fragment" or "TNFRI polypeptide fragment") refers to a fragment of TNFRI, so The fragment has an amino acid sequence that is 100% identical to the corresponding amino acid sequence of full-length TNFRI and exhibits a deletion of at least one amino acid residue of TNFRI. The deleted amino acid residue can be located anywhere in the polypeptide, such as N-terminal, C-terminal or in between. The fragment shares at least one biological property with full-length TNFRI. Representatives are fragments consisting of 105 or 126 or 171 amino acid sequences extending from amino acid residue 41 at the N-terminus of TNFRI, each fragment designated herein as TNFRI105, TNFRI126 and TNFRI171, respectively.

As used herein, the term "TNFRI variant" or "TNFRI mutant" or "TNFRI fragment variant", "TNFRI fragment mutant" or "modified TNFRI polypeptide" or "modified TNFRI polypeptide fragment" means the same as defined below TNFRI polypeptides or fragments thereof that have a sequence identity of less than 100% to TNFRI polypeptides or TNFRI fragments isolated from natural cells or recombinant cells. Typically, TNFRI mutants have about 70% or greater amino acid sequence identity to wild-type or native TNFRI or a fragment of TNFRI. Such sequence identity is preferably at least about 75%, more preferably at least about 80%, still more preferably at least about 85%, even more preferably at least about 90%, and most preferably at least about 95%.

As used herein, the term "quadruple mutant" refers to a mutant having mutations within 4 positions in the amino acid sequence of human tumor necrosis factor receptor-I or a fragment of human tumor necrosis factor receptor-I.

As used herein, the term "quintuple mutant" refers to a mutant having mutations within 5 positions in the amino acid sequence of human tumor necrosis factor receptor-I or human tumor necrosis factor receptor-I fragment.

As used herein, the term "sixfold mutant" refers to a mutant having mutations within 6 positions in the amino acid sequence of human tumor necrosis factor receptor-I or a fragment of human tumor necrosis factor receptor-I.

As used herein, the term "septamutant" refers to a mutant having mutations within 7 positions in the amino acid sequence of human tumor necrosis factor receptor-I or human tumor necrosis factor receptor-I fragment.

As used herein, the term "TNFRIm" refers to a fragment of TNFRI having an amino acid sequence consisting of m amino acids extending from amino acid residue 41 at the N-terminus of the amino acid sequence of TNFRI. For example, a TNFRI105 fragment refers to a TNFRI fragment having a 105 amino acid sequence extending from amino acid residue 41 of the terminus of TNFRIN. Another example is TNFRI126, which has a 126 amino acid sequence extending from amino acid residue 41 of the terminus of TNFRIN.

As used herein, the term "Met-TNFRIm" refers to a fragment of TNFRI having an amino acid sequence consisting of m amino acids extending from amino acid residue 41 at the terminal end of TNFRIN, wherein, for the purpose of expressing TNFRI in E. A methionine that was not originally present in the amino acid sequence of TNFRI was added to the N-terminus.

Amino acids appearing in the various amino acid sequences provided herein are identified according to known 3-letter or single-letter abbreviations. The nucleotides occurring in each nucleic acid fragment are designated by standard single-letter designations routinely used in the art.

The notation "xAz" as used herein refers to the substitution of amino acid x at position A by amino acid z in the amino acid sequence. For example, K48Q refers to a glutamine (Gln) residue replacing a lysine (Lys) residue at position 48.

The present invention relates to modified TNFRI polypeptides or fragments thereof having increased TNF-α binding capacity and improved protease resistance in vivo and/or in vitro, methods for their production and uses thereof.

The present inventors conducted a thorough and thorough study of TNFR1 mutants with improved TNF affinity and in vivo and/or in vitro stability, and obtained the following findings: 4 or more of the TNFRI sites within the expected TNF-binding site Substitution of amino acid residues leads to improved affinity for TNF, thereby realizing the present invention. However, because the resulting mutants with increased affinity for TNF are susceptible to enzymatic degradation, additional modifications that increase protease resistance were added to the mutants in order to select for mutants with protease resistance similar to or higher than native TNFRI.

Accordingly, the present invention provides modified TNFRI polypeptides or fragments thereof with improved TNF binding ability and protease resistance by substituting amino acids at 5 or more positions in specific positions of the native TNFRI amino acid sequence.

Due to the stable combination with TNF, the modified TNFRI polypeptide or its fragments of the present invention can effectively inhibit the action of TNF. Furthermore, since their activities are independent of sugar chain modification, they can be produced in microbial cells as well as animal cells.

A more detailed description of the present invention will be given below.

The present invention provides a modified tumor necrosis factor receptor-1 (TNFRI) or a fragment thereof, which comprises a modification of 5 amino acid residues, the 5 amino acid residues are represented by the wild-type TNFRI polypeptide shown in SEQ ID NO: 1 or its function In the amino acid sequence of the active fragment, 4 amino acid residues at positions 92, 95, 97 and 98 and 1 amino acid residue selected from one of positions 68, 161, 200 and 207 are composed. Therefore, the modified TNFRI The polypeptide or fragment has improved TNF binding ability compared to wild-type human tumor necrosis factor receptor-1 (TNFRI) polypeptide and protease resistance comparable to or higher than wild-type human tumor necrosis factor receptor-1 (TNFRI) polypeptide .

The present invention also provides a modified tumor necrosis factor receptor-1 (TNFRI) or a fragment thereof, except for the four amino acid residues at positions 92, 95, 97 and 98 and one selected from 68, 161, 200 and 207 thereof In addition to the modification of 5 amino acid residues consisting of 1 amino acid residue at one position, it also includes a further amino acid modification at position 93 in the amino acid sequence of the wild-type TNFRI polypeptide shown in SEQ ID NO: 1 or a functionally active fragment thereof.

Preferably, the present invention provides a modified tumor necrosis factor receptor-1 (TNFRI) or a fragment thereof, which comprises 68, 92, 95 of the amino acid sequence of the wild-type TNFRI polypeptide shown in SEQ ID NO: 1 or a functionally active fragment thereof , Modification of amino acid residues at positions 97 and 98. In addition, the present invention provides a modified tumor necrosis factor receptor-1 (TNFRI) or a fragment thereof, which, in addition to modification of amino acid residues at positions 68, 92, 95, 97 and 98, also includes the modification of SEQ ID NO: 1 Other modifications of the amino acid residue at position 161 or 207 in the amino acid sequence of the wild-type TNFRI polypeptide or its functionally active fragment.

The present invention provides a modified tumor necrosis factor receptor-1 (TNFRI) or a fragment thereof, which comprises a modification of 6 amino acid residues, the 6 amino acid residues being represented by the wild-type TNFRI polypeptide shown in SEQ ID NO: 1 or its The amino acid sequence of the functionally active fragment consists of four amino acid residues at positions 92, 95, 97 and 98 and two amino acid residues at two positions selected from positions 68, 161, 200 and 207.

Such modifications are intended to achieve improved binding to TNF and to ensure the same or higher protease resistance compared to wild-type TNFRI polypeptides or fragments thereof. Representative modifications are amino acid substitutions. However, any modification, including chemical modification of amino acid residues at the same position, such as post-translational modification, including the use of Glycosylation, acylation (eg, acetylation or succinylation), methylation, phosphorylation, hasylation, carbamylation, sulfation, prenylation, oxidation, guanidinylation, amidation of sugar moieties , carbamylation (eg, carbamoylation), trinitrobenzoation, nitration, and PEGylation.

In the case of amino acid substitution and modification of the amino acid sequence of the wild-type human tumor necrosis factor (TNFRI) polypeptide shown in SEQ ID NO: 1 or its functionally active fragment, the L at position 68 is replaced by V; by I, L, F, M, W, Q, T, Y, K, H, E, A, V, P, N or R replace S at position 92; E at position 93 is replaced by P; H at position 95 is replaced by F; Substitution of R at position 97 by P, L or I; substitution of H at position 98 by A or G; substitution of K at position 161 by Q or N; substitution of E at position 200 by Q; Bit of D. Preferably, S at position 92 is substituted by I, M or H; R at position 97 is substituted by P.

More preferably, the present invention provides modified human tumor necrosis factor receptor-1 or a fragment thereof, which is selected from the group consisting of L68V/S92I in the amino acid sequence of the wild-type human TNFRI polypeptide shown in SEQ ID NO: 1 or a functionally active fragment thereof /H95F/R97P/H98A, L68V/S92M/H95F/R97P/H98A, L68V/S92H/H95F/R97P/H98A, L68V/S92I/H95F/R97P/H98G, L68V/S92M/H95F/R97P/H98G, L68V/S92I /H95F/R97P/H98A/K161Q, L68V/S92I/H95F/R97P/H98A/K161N, L68V/S92I/H95F/R97P/H98A/D207N, L68V/S92M/H95F/R97P/H98A/K161Q, L68V/S92M/H9 /R97P/H98A/K161N, L68V/S92M/H95F/R97P/H98A/D207N, L68V/S92H/H95F/R97P/H98A/K161Q, L68V/S92H/H95F/R97P/H98A/K161N, L68V/S92H/H95F/R9 Amino acid modifications of /H98A/D207N, L68V/S92I/H95F/R97P/H98G/K161Q and L68V/S92M/H95F/R97P/H98G/K161N.

As used herein, a "functionally active fragment" of the wild-type human TNFRI polypeptide shown in the amino acid sequence of SEQ ID NO: 1 means a part of the wild-type human TNFR1 polypeptide that substantially performs the same function as the complete polypeptide. Specifically, the present invention utilizes an amino acid sequence consisting of amino acid residues 41-211 (SEQ ID NO:2; TNFRI171) of the natural human TNFRI amino acid sequence shown in SEQ ID NO:1; the natural human TNFRI amino acid sequence shown in SEQ ID NO:1 Amino acid sequence consisting of amino acid residues 41-166 (SEQ ID NO:3; TNFRI126) of amino acid residues 41-145 (SEQ ID NO:4; TNFRI105) of the natural human TNFRI amino acid sequence shown in SEQ ID NO:1. For reference, it is well known that a fragment extending from position 59 to position 143 in the amino acid sequence of human TNFRI polypeptide (SEQ ID NO: 1) exhibits biological activity of TNFRI (US Patent No. 6,989,147).

As the term is used herein, a "fragment" of a modified TNFRI polypeptide refers to a portion of the modified TNFRI polypeptide that has substantially the same effect as the modified TNFRI polypeptide and can be readily prepared by those skilled in the art.

Modified TNFR1 polypeptides and fragments thereof having improved affinity for TNF and having the same or improved resistance to proteases include those described below.

The present invention provides a modified human tumor necrosis factor receptor-1 polypeptide or a fragment thereof, which has amino acids consisting of amino acids 41-211 of the amino acid sequence of the wild-type human tumor necrosis factor receptor-1 polypeptide shown in SEQ ID NO:1 sequence, and the following amino acid substitutions: L at position 68 by V; substitution at position 92 by I, L, F, M, W, Q, T, Y, K, H, EA, V, P, N or R Substitution of S at position 95 by F; substitution of R at position 97 by P, L or I; and substitution of H at position 98 by A or G. More preferably, the present invention provides a modified human tumor necrosis factor receptor-1 polypeptide or a fragment thereof, which has amino acid 41- An amino acid sequence consisting of 211, and selected from the group consisting of L68V/S92I/H95F/R97P/H98A, L68V/S92M/H95F/R97P/H98A, L68V/S92H/H95F/R97P/H98A, L68V/S92I/H95F/R97P/H98G and L68V Modification of /S92M/H95F/R97P/H98G.

In addition, the present invention provides a modified human tumor necrosis factor receptor-1 polypeptide or a fragment thereof, which is composed of amino acids 41-211 of the amino acid sequence of the wild-type human tumor necrosis factor receptor-1 polypeptide shown in SEQ ID NO:1 and has, in addition to the above substitutions, provided the following amino acid substitutions: substitution of K at position 161 by Q or N, or substitution of D at position 207 by N. The modified human tumor necrosis factor receptor-1 polypeptide or its fragments of the present invention contain a polypeptide selected from L68V/S92I/H95F/R97P/H98A/K161Q, L68V/S92I/H95F/R97P/H98A/K161N, L68V/S92I/H95F/ R97P/H98A/D207N, L68V/S92M/H95F/R97P/H98A/K161Q, L68V/S92M/H95F/R97P/H98A/K161N, L68V/S92M/H95F/R97P/H98A/D207N, L68V/S92H/H95F/R97 H98A/K161Q, L68V/S92H/H95F/R97P/H98A/K161N, L68V/S92H/H95F/R97P/H98A/D207N, L68V/S92I/H95F/R97P/H98G/K161Q and L68V/S92M/H95F/R97P/H98 Modification of K161N.

The present invention provides a modified human tumor necrosis factor receptor-1 polypeptide or a fragment thereof, which has amino acids consisting of amino acids 41-166 of the amino acid sequence of the wild-type human tumor necrosis factor receptor-1 polypeptide shown in SEQ ID NO: 1 sequence, and the following amino acid substitutions: L at position 68 by V; substitution at position 92 by I, L, F, M, W, Q, T, Y, K, H, EA, V, P, N or R Substitution of S at position 95 by F; substitution of R at position 97 by P, L or I; and substitution of H at position 98 by A or G. More preferably, the present invention provides a modified human tumor necrosis factor receptor-1 polypeptide or a fragment thereof, which has amino acid 41- An amino acid sequence consisting of 166, and selected from the group consisting of L68V/S92I/H95F/R97P/H98A, L68V/S92M/H95F/R97P/H98A, L68V/S92H/H95F/R97P/H98A, L68V/S92I/H95F/R97P/H98G and L68V Modification of /S92M/H95F/R97P/H98G.

In addition, the present invention also provides a modified human tumor necrosis factor receptor-1 polypeptide or a fragment thereof, which has amino acids 41-166 of the amino acid sequence of the wild-type human tumor necrosis factor receptor-1 polypeptide shown in SEQ ID NO:1 and, in addition to the above substitutions, also has an amino acid substitution of Q or N substituting K at position 161. The modified human tumor necrosis factor receptor-1 polypeptide of the present invention or its fragments contain a protein selected from L68V/S92I/H95F/R97P/H98A/K161Q, L68V/S92I/H95F/R97P/H98A/K161N, L68V/S92M/H95F/ R97P/H98A/K161Q, L68V/S92M/H95F/R97P/H98A/K161N, L68V/S92H/H95F/R97P/H98A/K161Q, L68V/S92H/H95F/R97P/H98A/K161N, L68V/S92I/H95F/R97 Modifications of H98G/K161Q and L68V/S92M/H95F/R97P/H98G/K161N.

The present invention provides a modified human tumor necrosis factor receptor-1 polypeptide or a fragment thereof, which has amino acids consisting of amino acids 41-145 of the amino acid sequence of the wild-type human tumor necrosis factor receptor-1 polypeptide shown in SEQ ID NO:1 sequence with the following amino acid substitutions: L at position 68 replaced by V; replaced by I, L, F, M, W, Q, T, Y, K, H, E, A, V, P, N or R S at position 92; H at position 95 by F; R at position 97 by P, L or I; and H at position 98 by A or G. More preferably, the present invention provides a modified human tumor necrosis factor receptor-1 polypeptide or a fragment thereof, which has amino acid 41- 145, and has an amino acid sequence selected from L68V/S92I/H95F/R97P/H98A, L68V/S92M/H95F/R97P/H98A, L68V/S92H/H95F/R97P/H98A, L68V/S92I/H95F/R97P/H98G and Modification of L68V/S92M/H95F/R97P/H98G.

In addition, the present invention provides a modified human tumor necrosis factor receptor-1 polypeptide or a fragment thereof, which has an amino acid sequence that is at least 90%, at least 95%, at least 96%, An amino acid sequence having at least 97%, at least 98% or at least 99% sequence homology, with modifications corresponding to positions 68, 92, 95, 97 and 98 of the amino acid sequence of SEQ ID NO:1. In one embodiment, the modification is the following amino acid substitutions at positions corresponding to positions 68, 92, 95, 97 and 98 of the amino acid sequence of SEQ ID NO: 1: replacing L by V; replacing L by I, L, F, M, W, Q, T, Y, K, H, E, A, V, P, N, or R for S; F; R for P, L, or I; and A or G for H. The modified human tumor necrosis factor polypeptide or a fragment thereof may also comprise substitution of K by Q or N; or substitution of D by N at positions corresponding to positions 161 and 207 of the amino acid sequence of SEQ ID NO:1.

In addition, the present invention also provides a modified TNFRI polypeptide or a fragment thereof, which has substantially the same amino acid sequence as SEQ ID NO: 1, and the above-mentioned or corresponding amino acid modifications introduced therein. As used herein, the term "polypeptide having substantially the same amino acid sequence as SEQ ID NO: 1" means a TNFRI polypeptide with amino acid modifications, such as amino acid substitutions, deletions, additions, in a number and type that do not reduce its intrinsic TNFRI activity. More specifically, the invention provides modified TNFRI polypeptides or fragments thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid sequence of SEQ ID NO:1 Homologous amino acid sequences with modifications corresponding to the amino acid modifications described above.

In addition to the above-mentioned amino acid modification of the present invention, the above-mentioned variant has more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 98%, or greater than 99% sequence homology, and include allelic variant isoforms, tissue-specific isoforms, and allelic variants thereof of human TNFRI polypeptides, with one or more amino acid mutations, substitutions, deletions, Inserted or added synthetic variants, synthetic molecules prepared by translation of nucleic acids, proteins isolated from human and non-human tissues and cells, chimeric TNFRI polypeptides and modified forms thereof.

As used herein, the term "corresponding modification" refers to a modification of a residue in or between polypeptides of other isoforms compared. That is, "corresponding modification" means that at a position having a residue that is functionally unalterable by sequence alignment with the amino acid sequence of the natural human TNFRI polypeptide shown in SEQ ID NO: 1, it corresponds to the residue used to improve the binding ability and Modifications corresponding to amino acid modifications of the invention that maintain or improve protease resistance. Those skilled in the art can readily identify corresponding residue modifications between or within such polypeptides. For example, those skilled in the art can identify corresponding residues by aligning the sequences of TNFRI polypeptides using conserved and identical amino acid residues as a guide.

Preferably, the present invention provides any one comprising SEQIDNOS: 6, 11, 16, 21, 22, 28-39, 44, 49, 54, 55, 61-72, 75, 78, 81, 82 or 86-98 TNFRI polypeptides of the amino acid sequences shown or fragments thereof.

In addition to the above amino acid modifications, for the purpose of increasing protease resistance, reducing immunogenicity, or maintaining or enhancing biological activity, the modified TNFRI polypeptide or fragment thereof of the present invention may also contain other chemical modifications, such as protein translation Post-modification, e.g., glycosylation, acylation (e.g., acetylation or succinylation), methylation, phosphorylation, hesylation, carbamylation, sulfation, prenylation, oxidation, Guanidinylation, amidation, carbamylation (eg, carbamoylation), trinitrobenzolation, nitration, and PEGylation.

According to another aspect, the invention provides a multimeric polypeptide (or "polypeptide complex") comprising two or more copies of a modified human TNFRI polypeptide or a fragment thereof.

In addition, the present invention provides genes encoding the aforementioned TNFRI polypeptides or fragments thereof.

Genes encoding TNFRI polypeptides of the invention or fragments thereof include genes engineered for optimal expression in E. coli. When human genes are expressed in E. coli, the expression yield of the genes is low due to the difference in gene codons between humans and E. coli. For this reason, a human TNFRI gene-based gene engineered for expression in E. coli, such as the TNFRI gene of SEQ ID NO: 5, can be used in the present invention. When inserted into an E. coli expression vector (e.g., pET44a (Cat. No.: 71122-3, Novagen)) and then expressed in E. coli cells without codon additions (e.g.: BL21(DE3)), this gene showed a higher Higher expression levels of the human TNFRI gene. Therefore, using the above-mentioned genes, TNFRI fragments and TNFRI mutants can be efficiently produced in Escherichia coli.

In addition, the present invention also provides a vector containing the gene. In the present invention, the vectors that can be used to introduce genes can be vectors known in the art, preferably vectors with the cleavage map shown in FIG. 1 .

In addition, the present invention provides cells (microbial cells or animal cells) transformed with such vectors. In the present invention, microbial cells or animal cells that can be used for transformation vectors can be known microbial cells or animal cells used for transformation in the art, preferably Escherichia coli cells, CHO cells or HEK293 cells, and more preferably Escherichia coli cells (eg, E. coli BL21(DE3)).

The present invention provides methods for producing TNFRI using E. coli.

TNFRI can be produced by using animal cells (Bernie et al., The Journal of Pharmacology and Experimental Therapeutics. 301:418-426, 2002; and Scallon et al., Cytokine. 7:759-770, 1995).

Since TNFRI is expressed as a conformationally inactive inclusion body when expressed in E. coli, refolding into an active protein is required (Silvia et al., Analytical Biochemistry 230:85-90, 1995; and Karin et al., Cytokine. 7:26-38, 1995). Accordingly, the modified TNFRI polypeptides or fragments thereof of the present invention can be produced by expressing TNFRI in the form of inclusion bodies in E. coli, refolding the expressed TNFRI into active TNFRI, and by using gel filtration chromatography, etc. Active TNFRI is purified. Alternatively, the modified TNFRI polypeptide or fragment thereof of the present invention can be produced in Escherichia coli in the form of a soluble protein instead of an inclusion body, using an expression method involving the linkage of a hydrophilic fusion protein, a low-temperature culture method, and the like. For example, TNFRI was produced as a soluble protein in E. coli by linking the hydrophilic NusA protein to the N-terminus of the TNFRI protein.

In addition, the present invention provides a method for producing a TNFRI polypeptide or a fragment thereof, the method comprising introducing the gene into a suitable vector, transforming the vector into a host cell to produce a transformant, and in a culture medium Transformants are grown to express a TNFRI polypeptide or fragment thereof.

In addition, the present invention provides a method for treating a TNF-mediated disease or medical condition (hereinafter referred to as "TNF-mediated disease"). Examples of TNF-mediated diseases, sequelae associated therewith, and symptoms associated therewith include: adult respiratory distress syndrome; anorexia; cancer (e.g., leukemia); chronic fatigue syndrome; graft-versus-host rejection; hyperalgesia; Inflammatory bowel disease; neuroinflammatory disease; ischemia/reperfusion injury, including cerebral ischemia (brain injury due to trauma, epilepsy, hemorrhage, or stroke, each of which can lead to neurodegeneration); diabetes ( eg, juvenile-onset type 1 diabetes); multiple sclerosis; eye disease; pain; pancreatitis; pulmonary fibrosis; rheumatic diseases (eg, rheumatoid arthritis, osteoarthritis, juvenile (rheumatoid) Negative polyarthritis, ankylosing spondylitis, Reiter syndrome and reactive arthritis, psoriatic arthritis, enteropathic arthritis, polymyositis, dermatomyositis, scleroderma, systemic sclerosis, vasculitis, cerebral vasculitis, Sjogren's syndrome, rheumatic fever, polychondritis and polymyalgia, rheumatoid arteritis, and giant cell arteritis); septic shock; side effects from radiation therapy; systemic lupus erythematosus; temporal Jaw joint disease; thyroiditis and tissue transplantation. TNF-mediated diseases are well known in the art.

In addition, the present invention provides a composition for preventing or treating rheumatoid arthritis or TNF-mediated diseases, which contains a modified TNFRI polypeptide or a fragment thereof.

In addition, the present invention provides a composition for preventing or treating rheumatoid arthritis or TNF-mediated diseases, which contains a gene encoding a modified TNFRI polypeptide or a fragment thereof, a vector containing the gene, or a Microbial cells or animal cells transformed with the above-mentioned vectors.

In addition, the present invention provides a method for preventing or treating rheumatoid arthritis or a TNF-mediated disease, the method comprising administering to a subject in need thereof a method for preventing or treating TNF-mediated Composition of diseases.

The pharmaceutical compositions of the present invention may be administered orally, sublingually, rectally, transdermally, subcutaneously, intramuscularly, intraperitoneally, intravenously or intraarterially. The pharmaceutical composition can be prepared for storage or administration by mixing the TNFRI mutant having the desired purity with a pharmaceutically acceptable carrier, excipient or stabilizer. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, acetates and other organic acid salts; antioxidants such as ascorbic acid; Low molecular weight (fewer than about 10 residues in length) peptides, including polyarginine and proteins, such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine acid, aspartic acid, or arginine; and other sugars, including monosaccharides, disaccharides, cellulose and its derivatives, glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or Sorbitol; counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).

The pharmaceutical composition of the present invention can be formulated into a sterile composition for injection according to conventional methods known in the art. Sterile compositions for injection may contain solutions or suspensions of the active compounds in vehicles such as water, or naturally occurring vegetable oils such as sesame oil, peanut oil, or cottonseed oil, or synthetic lipid vehicles such as ethyl oleate. Sterile compositions for injection may incorporate buffers, preservatives, antioxidants and the like according to conventional pharmaceutical practice.

The modified TNFRI polypeptide of the present invention or its fragment, or the gene encoding said polypeptide or its fragment, the vector containing said gene, or the microbial cell or animal cell transformed with said vector to treat TNF-mediated disease A therapeutically effective amount is incorporated into the pharmaceutical composition.

As used herein, the term "therapeutically effective amount" refers to the amount/dose of an active ingredient or pharmaceutical composition sufficient to produce an effective response in a subject after administration (i.e., the researcher, veterinarian, physician or other clinician seeks to animal or human biological or medical response). A therapeutically effective amount is intended to include an amount that causes alleviation of the symptoms of the disease or condition being treated. It will be apparent to those skilled in the art that the therapeutically effective amount and frequency of administration of the active ingredients of the present invention will vary depending on the desired effect. Therefore, those skilled in the art can easily determine the optimal dose and can be based on various factors, such as the type and severity of the disease, the content of the active ingredient and other ingredients in the composition, the dosage form, and the subject's age, body weight, Physical condition and sex, as well as diet, timing and route of administration and rate of excretion of the composition, duration of treatment and concomitant drugs are adjusted. For example, for adults, the TNFRI mutants of the present invention are preferably administered at a dose of 0.01 to 1.000 mg/kg once a day and more preferably 0.1 to 100 mg/kg once a day.

The TNFRI polypeptides or fragments thereof of the invention may be administered adjunctively to other therapies and may be administered with other pharmaceutical compositions appropriate for the indication being treated. The TNFRI polypeptides or fragments thereof of the invention can be administered alone or in combination with any of one or more known or novel anti-inflammatory drugs. Information on compounds corresponding to this class of drugs can be found in "The Merck Manual of Diagnosis and Therapy", 16th Edition, Merck, Sharp & Dohme Research Laboratories, Merck & Co., Rahway, N.J. (1992) and "Pharmaprojects", PJB Publications Ltd.

As an example of combined use, the TNFRI polypeptides of the present invention or fragments thereof can be used in combination with first-line drugs for controlling inflammation and classified as non-steroidal anti-inflammatory drugs (NSAIDs) for the treatment of TNF-mediated diseases, including acute and Chronic inflammation, such as rheumatic diseases (eg, Lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, and staphylococcal ("septic") joints inflammation).

As another example of combined use, the TNFRI polypeptide of the present invention or a fragment thereof can be combined with any one or more long-acting antirheumatic drugs (SAARD) or disease-modifying antirheumatic drugs (DMARD), its prodrug ester or druggable Combinations with salts are useful in the treatment of TNF-mediated diseases as defined above and multiple sclerosis.

As yet another example of combined use, the TNFRI polypeptides of the present invention or fragments thereof can be used in combination with any one or more COX2 inhibitors, prodrug esters or pharmaceutically acceptable salts thereof for the treatment of TNF-mediated diseases as defined above .

In addition, the TNFRI polypeptides or fragments thereof of the present invention may be used in combination with any one or more antibacterial drugs, prodrug esters or pharmaceutically acceptable salts thereof for the treatment of TNF-mediated diseases as defined above.

TNFRI polypeptides of the present invention or fragments thereof may be used in combination with any one or more of the compounds given below for the treatment of TNF-mediated diseases as defined above: granulocyte colony-stimulating factor; thalidomide; tenida Tiapafant; Nimesulide; Panavir; Rolipram; Sulfasalazine; Balsalazide; Osalazine; Mesalazine; Prednisolone; Budesonide; Methylprednisolone ; hydrocortisone; methotrexate; cyclosporine; peptide T; (1R,3S)-cis-1-[9-(2,6-diaminopurinyl)]-3-hydroxy-4- Cyclopentene hydrochloride; (1R,3R)-trans-1-[9-(2,6-diamino)purine]-3-acetoxycyclopentane; (1R,3R)-trans-1- [9-Adeninyl)-3-azidocyclopentane hydrochloride and (1R,3R)-trans-1-[6-hydroxy-purin-9-yl)-3-azidocyclopentane .

The TNFRI polypeptides or fragments thereof of the invention may be used in combination with one or more additional TNF inhibitors for the treatment of TNF-mediated diseases as defined above. Such TNF inhibitors include compounds and proteins that block the in vivo synthesis or extracellular release of TNF: for example, anti-TNF antibodies, including MAK195FFab antibody (Holler et al., (1993), 1st International Symposium on Cytokines in Bone Marrow Transplantation, 147); CDP571 anti-TNF monoclonal antibody (Rankin et al., (1995), British Journal of Rheumatology, 34:334-342); BAYX1351 mouse anti-TNF monoclonal antibody (Kieft et al., (1995), 7th European Congress of Clinical Microbiology and Infectious Diseases, 9); CenTNFcA2 anti-TNF monoclonal antibody (Elliott et al. al., (1994), Lancet, 344:1125-1127 and Elliott et al., (1994), Lancet, 344:1105-1110).

Additionally, the present invention provides pharmaceutical preparations comprising TNFRI polypeptides or fragments thereof. Preferably, the pharmaceutical preparations according to the invention also contain pharmaceutically acceptable excipients. The pharmaceutical preparation of the present invention may be in the form of a pharmaceutical preparation selected from oral preparations, inhalers, injections, transmucosal preparations and external use.

The pharmaceutical preparations of the present invention contain therapeutically effective amounts of pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants or carriers.

In addition, the pharmaceutical product of the present invention contains additives commonly used in the art, including buffers (such as Tris buffer, acetate buffer or phosphate buffer), detergents (such as Tween80), antioxidants (such as ascorbic acid, pyrolysis, etc.) sodium sulfite), preservatives (eg, thimerosal, benzyl alcohol), and filler substances (eg, lactose, mannitol). Additives can be incorporated into particulate preparations of polymers such as polylactic acid or polyglycolic acid or into liposomes. The pharmaceutical preparations according to the invention may contain hyaluronic acid for the purpose of promoting long-lasting maintenance in circulation. The pharmaceutical preparations of the present invention may optionally contain pharmaceutically acceptable liquid, semi-solid or solid diluents that serve as pharmaceutical vehicles, excipients or vehicles, including but not limited to polyoxyethylene sorbitan monolaurate, starch , Sucrose, Dextrose, Gum Arabic, Calcium Phosphate, Mineral Oil, Cocoa Butter and Cocoa Butter.

The pharmaceutical preparations according to the invention also contain inert additives which provide protection against the gastric environment and release of the biologically active substance in the intestine.

The pharmaceutical preparations of the invention are prepared using known techniques including mixing, dissolving, granulating, dragging, levigating, emulsifying, encapsulating, entraining or tabletting methods.

The pharmaceutical preparations of the present invention may be in liquid (e.g. suspensions, elixirs and/or solutions) or solid (e.g. powders, tablets and/or capsules) form, or may be formulated as depots form. Depot formulations generally have a longer duration of action than non-depot formulations. Depot formulations may be prepared using suitable polymeric or hydrophobic materials (for example, as emulsions in suitable oils) or ion exchange resins, or as sparingly soluble derivatives, for example, as sparingly soluble salts. In addition, the pharmaceutical preparations according to the invention contain delivery systems, such as liposomes or emulsions. Certain delivery systems are used in the preparation of certain pharmaceutical products, including those containing hydrophobic compounds. In certain embodiments, organic solvents such as dimethyl sulfoxide are used. In another aspect of the invention, the pharmaceutical preparations of the invention contain one or more tissue-specific delivery molecules designed to deliver an agent to a particular tissue or cell type. For example, in certain embodiments, pharmaceutical preparations contain liposomes coated with tissue-specific antibodies.

Preferably, the pharmaceutical preparations of the present invention may be formulated as oral solid dosage forms. Solid dosage forms include tablets, capsules, pills, troches or pellets.

Additionally, liposomal or proteinoid encapsulation methods may be used to formulate the compositions of the present invention. Liposomes can be prepared from phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), and sphingomyelin (SM); and hydrophilic polymers, such as Polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polyacrylamide, polydimethylacrylic Amide, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethyl cellulose, hydroxyethyl cellulose, polyethylene glycol and polyasparagine.

If desired, the TNFRI polypeptides or fragments thereof contained in the pharmaceutical preparations of the present invention may be chemically modified for effective oral delivery. Typically, the chemical modification involved is the attachment of at least one moiety to a TNFRI mutant polypeptide, wherein said moiety may confer protease resistance or facilitate uptake of a substance from the stomach or intestine into the bloodstream. Preferably, the moiety for chemical modification may be a moiety for chemical modification which increases the overall stability of the pharmaceutical product of the invention and thus increases its circulation time in the body. Examples of such moieties include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone and polyproline. Other polymers that can be used are poly-1,3-dioxane and poly-1,3,6-trioxane. Most preferred are polyethylene glycol moieties (PEGylated).

As a carrier to enhance absorption of the pharmaceutical product of the present invention in an oral dosage form, salts of modified aliphatic amino acids, such as sodium N-(8-[2-hydroxybenzoyl]amino)caprylate (SNAC) may be used.

The pharmaceutical preparations of the invention can be formulated as fine multiparticulates in the form of granules or agglomerates with a particle size of about 1 mm. In this case, the drug can be in the form of capsules. Multiparticulate preparations may be in the form of powders, lightly compressed plugs or tablets. Such formulations can be prepared by compression.

The pharmaceutical preparations of the present invention can also be formulated in the form of liposome or microsphere encapsulation, further incorporating coloring agents and flavoring agents.

In addition, in order to enhance the uptake of TNFRI polypeptides or fragments thereof as active ingredients in the pharmaceutical preparations of the present invention, additives including fatty acids such as oleic acid or linoleic acid may be used.

The pharmaceutical preparations of the invention may be controlled release formulations. TNFRI polypeptides or fragments thereof, as active ingredients in such formulations, may be incorporated into inert carriers that allow controlled release by diffusion or dissolution mechanisms. In addition, controlled release formulations may contain slowly disintegrating matrices, for example, alginates or polysaccharides. Another form of controlled release formulation may be based on an osmotic release oral delivery system (OROS, AlzaCorp.). In the controlled-release formulation, the TNFRI mutant as the active ingredient of the present invention is enclosed in a semipermeable membrane that allows water to enter and pushes the active ingredient out through a single small opening due to osmotic effect. The controlled-release formulation of the present invention may have an enteric coating to exhibit a delayed-release effect of the drug.

The pharmaceutical preparations of the invention may be in the form of film-coated tablets. Materials used in film coating fall into two categories. The first class is non-enteric materials and includes methylcellulose, ethylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, carboxy Sodium methylcellulose, povidone, and polyethylene glycol. The second category includes enteric materials such as esters of phthalic acid. Specifically, the enteric polymer used as the enteric material is selected from enteric cellulose derivatives, enteric acrylic acid copolymers, enteric maleic acid copolymers, enteric polyvinyl derivatives and combinations thereof. The enteric cellulose derivative is at least one selected from the group consisting of hypromellose acetate succinate, hypromellose phthalate, hydroxymethylethylcellulose phthalate , Cellulose acetate phthalate, Cellulose acetate succinate, Cellulose acetate maleate, Cellulose benzoate phthalate, Cellulose propionate phthalate, Methyl Cellulose phthalate, carboxymethyl ethyl cellulose and ethyl hydroxyethyl cellulose phthalate. The enteric acrylic acid copolymer is at least one selected from the following: styrene-acrylic acid copolymer, methyl acrylate-acrylic acid copolymer, acrylic acid-methyl methacrylate copolymer, butyl acrylate-styrene-acrylic acid copolymer, Methacrylic acid-methyl methacrylate copolymers (for example, Eudragit L100, Eudragit S, Degussa), methacrylic acid-ethyl acrylate copolymers (for example, Eudragit L100-55, Degussa) and methyl acrylate-methacrylic acid-octyl acrylate ester copolymer. The enteric maleic acid copolymer is at least one selected from the following: vinyl acetate-maleic anhydride copolymer, styrene-maleic anhydride copolymer, styrene-maleic acid monoester copolymer, vinyl methyl Ether-maleic anhydride copolymer, ethylene-maleic anhydride copolymer, vinyl butyl ether-maleic anhydride copolymer, acrylonitrile-methyl acrylate-maleic anhydride copolymer and butyl acrylate-styrene-maleic anhydride anhydride copolymer. The enteric polyvinyl derivative is at least one selected from the following: polyvinyl alcohol phthalate, polyvinyl acetal phthalate, polyvinylbutyrate phthalate (polyvinylbutyratephthalate) and polyvinylacetacetalphthalate.

Mixtures of the above coating materials may be used to provide optimum film coatings. The film coating process can be carried out in a coating pan or in a fluid bed granulator or by a compression coater.

For the purpose of sustained drug release, the controlled release pharmaceutical preparations of the invention may contain the TNFRI polypeptides of the invention or fragments thereof in a semipermeable matrix of solid hydrophobic polymers in the form of shaped articles (eg, films or microcapsules). Examples of sustained release matrices include polyesters, hydrogels [for example, as Langer et al., J.Biomed.Mater.Res., 15:167-277, 1981 and Langer, Chem.Tech., 12:98-105, 1982] poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)], polylactic acid (US Patent No. 3,773,919, EP58,481), L-glutamic acid and γ-ethyl-glutamine Copolymers of amino acid esters, non-degradable ethylene-vinyl acetate (Langer et al., supra), degradable lactic-co-glycolic acid (e.g., Lupron DepotTM (composed of lactic-co-glycolic acid and leuprolide Injectable microspheres composed of acetate) and poly-D-(-)-3-hydroxybutyrate (EP133,988).

When encapsulated proteins remain in the body for long periods of time, they may denature or aggregate due to exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Rational strategies for stabilizing proteins can be devised depending on the mechanisms involved. For example, if the mechanism of aggregation is found to be intermolecular S-S bond formation due to disulfide exchange, it can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific combinations of polymer matrices. material to achieve stabilization.

In addition, the present invention also provides the use of the TNFRI mutant of the present invention and pharmaceutical preparations containing the mutant. Such pharmaceutical preparations may be administered by injection or by oral, nasal, transdermal or other administration, including, for example, by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, intraocular, intrapulmonary or subcutaneous Injection; sublingual, anal, vaginal, or by surgical implant. Treatment may consist of a single dose or of multiple doses given over time.

Additionally, the pharmaceutical preparations of the present invention may be delivered by pulmonary delivery methods. The pharmaceutical product of the present invention is delivered to the lungs of a mammal while being inhaled, and it traverses the lung epithelium into the bloodstream.

Various types of mechanical devices designed for pulmonary drug delivery can be used for pulmonary delivery of the pharmaceutical products of the present invention. Examples of such devices include nebulizers, metered dose inhalers and powder inhalers, all of which are commercially available in the art.

The pharmaceutical preparations of the present invention may be suitably formulated for optimal use in the aforementioned devices. In general, each formulation is specific to the type of device employed and may involve the use of suitable propellants, in addition to diluents, adjuvants or carriers useful in therapy.

Pharmaceutical preparations of the invention for pulmonary delivery are preferably provided in granular form having an average particle size of about 10 μm or less, most preferably about 0.5 to 5 μm for efficient delivery to the distal lung.

Pharmaceutical preparations of the invention for pulmonary delivery may also contain sugars such as trehalose, mannitol, xylitol, sucrose, lactose or sorbitol as carriers. The pharmaceutical preparation may also contain dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) and dioleoylphosphatidylcholine (DOPC). Such pharmaceutical preparations may also contain natural or synthetic surfactants. Such pharmaceutical preparations may also contain polyethylene glycol, dextran such as cyclodextran, cholic acid and other related derivatives and amino acids used in buffered formulations.

Additionally, liposomes, microcapsules or microspheres, clathrates, or other types of carriers may also be used for pulmonary delivery of the pharmaceutical preparations of the invention.

Pulmonary delivery of the pharmaceutical products of the invention can be effected by jet or ultrasonic means using a nebulizer. A pharmaceutical product of the invention suitable for use in a nebulizer contains the TNFRI mutant dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. Nebuliser formulations may also contain buffers and monosaccharides that contribute, for example, to protein stabilization and regulation of osmotic pressure. Nebuliser formulations may also contain surfactants to reduce or prevent surface-induced protein aggregation caused by nebulization of the solution when forming an aerosol.

Pharmaceutical preparations of the invention for use with metered-dose inhaler devices will generally contain a powder of the finely divided composition comprising the TNFRI mutant of the invention suspended in a propellant by means of a surfactant . The propellants may be chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons or hydrocarbons, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethane and 1,1,1,2-tetrafluoro Ethane or combinations thereof. Examples of suitable surfactants that can be used herein include sorbitan trioleate and soy lecithin. Oleic acid may also be used as a surfactant.

Pharmaceutical preparations of the invention for dispensing from a powder inhalation device consist of a finely divided dry powder of a composition comprising a TNFRI mutant of the invention, and may also contain fillers such as lactose, sorbitol, sucrose, mannitol, trehalose or Xylitol. These can facilitate dispersion of the powder from the device.

The pharmaceutical preparations of the invention may also be delivered nasally. Nasal delivery allows the protein therapeutic to reach the bloodstream directly after administration of the protein therapeutic to the nose, thus preventing pulmonary deposition of the therapeutic product. Pharmaceutical preparations of the present invention for nasal delivery contain dextran or cyclodextran or the like. The pharmaceutical preparations of the invention may also be delivered across other mucosal membranes.

Dosage regimens for the pharmaceutical preparations of the present invention involved in methods of treatment of the above-mentioned diseases or conditions may be determined by the attending physician on the basis of various factors regulating the action of the drug, such as the age, condition, weight, sex and diet of the patient, the severity of any infection, Timing of administration and other clinical factors.

The pharmaceutical preparations of the invention may be administered by single or continuous administration, but are preferably administered by an initial bolus followed by continuous infusion in such a way as to maintain therapeutic levels of drug in circulation. Effective dosages and administration regimens can be easily optimized by common techniques known in the art. The frequency of dosing will depend on the pharmacokinetic parameters of the agent and the route of administration. Dosage regimens, administration regimens and frequency of administration can also be optimized based on the physical state, stability, rate of in vivo release and rate of in vivo clearance of the administered agent. For each route of administration, appropriate dosages can be calculated based on body weight, body surface area or organ size. Appropriate dosages may be ascertained by well established assays for determining dosages in blood levels, along with appropriate dose-response data. The final dosage regimen will be determined by the attending physician based on various factors that mediate the drug's action, such as drug specific activity, severity and patient responsiveness, patient's age, condition, weight, sex, and diet, and other clinical factors. As studies are conducted, additional information regarding appropriate dosage levels and duration of treatment for various diseases and conditions will emerge.

Beneficial effect

Compared with wild-type human tumor necrosis factor receptor-1 polypeptide or fragment thereof, the modified human tumor necrosis factor receptor-1 polypeptide or fragment thereof of the present invention has improved ability to bind to TNF, and equal or higher Protease resistance. Owing to these advantages over wild-type polypeptides, the modified polypeptides of the present invention show increased half-life in vivo and ensure improved bioavailability and absorption rates upon oral administration or injection. Therefore, the modified human tumor necrosis factor receptor-1 polypeptide or fragment thereof of the present invention can be advantageously used as an active ingredient in long-acting injections or oral preparations.

Description of drawings

Fig. 1 is a schematic diagram illustrating the construction of a Met-TNFRI105, Met-TNFRI126 or Met-TNFRI171 expression vector for Escherichia coli by inserting the Met-TNFRI105, Met-TNFRI126 or Met-TNFRI171 gene into pET44a.

Figure 2A is a gel filtration chromatogram showing the eluted Met-TNFRI105, Met-TNFRI126 and Met-TNFRI171 proteins (fraction A4), and Figure 2B is a graph showing purified Met- Photographs of TNFRI105, Met-TNFRI126 and Met-TNFRI171.

Fig. 3 is a graph showing the binding ability of TNFRI fragments (TNFRI126, TNFRI171) and TNFRI mutant fragments (TNFRI126-A30, TNFRI171-A30) to bind TNF-α as analyzed by ELISA.

Figure 4A is a graph showing the biological activity of TNFRI fragments (TNFRI126, TNFRI171) and TNFRI fragment mutants (TNFRI126-A30, TNFRI171-A30) as shown by the neutralization analysis of cytotoxicity against TNF-α; Figure 4B is a graph showing the biological activity of ENBREL ^™ , TNFRI fragments (TNFRI171) and TNFRI fragment mutants (TNFRI171-A2, TNFRI171-A9) shown by the neutralization of cytotoxicity against TNF-α; and Figure 4C is Graph showing biological activity of ENBREL ^™ , TNFRI fragment (TNFRI171) and TNFRI fragment mutants (TNFRI171-A21, TNFRI171-A22) as indicated by neutralization of cytotoxicity against TNF-α.

Fig. 5 is a schematic diagram illustrating the construction of a TNFRI108 expression vector for Escherichia coli by inserting the TNFRI108 gene into pET44a carrying the NusA gene.

Fig. 6 is a photograph showing expression of NusA-fused TNFRI108 protein in Escherichia coli transformed with pET44a-NusA-TNFRI108 expression vector, followed by purification by immobilized metal affinity chromatography and hydrophobic interaction chromatography.

Figure 7 is a graph showing protease resistance of TNFRI108 fragments and TNFRI108 single mutants TNFRI108-R14, TNFRI108-R64 and TNFRI108-R68.

Fig. 8 is a graph showing protease resistance of Met-TNFRI171 fragments and Met-TNFRI171 single mutants Met-TNFRI171-R83, Met-TNFRI171-R84 and Met-TNFRI171-R92.

Figure 9A is a graph showing the protease resistance of TNFRI171 fragments and TNFRI171 fragment mutants TNFRI171-A2, TNFRI171-S31 and TNFRI171-S53, and Figure 9B is a graph showing TNFRI171 fragments and TNFRI171 fragment mutants TNFRI171-A22, TNFRI171-S47 and TNFRI171 - Graph of protease resistance of S63.

Figure 10A is a graph showing the in vivo therapeutic effect of TNFRI171 fragment mutants on carrageenan-induced paw edema in mice, and Figure 10B is a graph showing IL-6 levels in edema-causing paw tissue (from left to right , Excipient, Enbrel, TNFRI171-S54, TNFRI171-S62, TNFRI171-A2, TNFRI171-A9, TNFRI171-S36, TNFRI171-S57, TNFRI171-S58 and TNFRI171-S63).

Figure 11A is a graph showing the in vivo therapeutic effect of TNFRI171 fragments and TNFRI171 fragment mutants on carrageenan-induced paw edema in mice, and Figure 11B is a graph showing IL-6 levels in paw tissues causing edema (from left To the right, Excipient, Enbrel, WT2.5mg/kg, WT5mg/kg, TNFRI-S6 32.5mg/kg, TNFRI-S635mg/ml).

detailed description

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following embodiments. However, the present invention is not limited to the Examples disclosed below.

The modified TNFRI polypeptides or fragments thereof of the present invention are prepared using information on the human TNFRI genome whose genome has been publicly disclosed.

[Preparation Example 1] Construction of TNFRI gene fragments and vectors for expression

(1) Construction of TNFRI171 gene fragment

It has been reported that human TNFRI has 4 extracellular domains, the binding of TNFRI to TNF-α is still possible even with only 3 domains of TNFRI (TNFRI126), and the absence of more of these extracellular domains does not affect the binding of TNFRI to TNF-α. Binding of TNF-alpha. Based on this fact, TNFRI171 having 171 amino acid residues extending from amino acid residue 41 of the human TNFRI polypeptide as described in SEQ ID NO: 1, TNFRI171 having 126 amino acid residues extending from amino acid residue 41 of human TNFRI polypeptide were selected. TNFRI126 of human TNFRI, and TNFRI105 with 105 amino acid residues extending from amino acid residue 41 of human TNFRI were used as candidate peptides for constructing the mutants of the present invention. To generate such mutants, the nucleotide sequence of TNFRI171 (SEQ ID NO: 5) was modified to facilitate expression in E. coli using codons matching E. coli. This sequence was constructed by PCR-based gene synthesis methods.

In order to insert the synthetic gene sequence into the pGEM-T (catalog number: A1380, Promega) vector, 3 μl of the synthetic gene was added to 1 μl of the pGEM-T vector, and 1 μl of ligase (catalog number: M2200S, NEB) was added thereto and ligation solution (2x ligation buffer), followed by reaction at room temperature for 10 minutes. Remove 2 μl of the reaction solution and add to XL1-blue competent cells (catalogue number: RH119-J80, RBC), which are then transformed by heat shock at 37°C for 2 minutes, followed by LB solid medium containing ampicillin cultured statically to obtain colonies. Colonies were cultured in LB liquid medium containing ampicillin, from which plasmids were isolated and gene sequences were confirmed by the ddNTP fluorescent labeling method using PCR (SolGent Inc., South Korea). This gene was named pGEM-TNFRI171. Thereafter, using the pGEM-TNFRI171 gene obtained above as a template, TNFRI126 and TNFRI105 genes were obtained by PCR.

(2) Construction of TNFRI expression vectors: Construction of TNFRI105, TNFR126 and TNFRI171 expression vectors

The commercially available vector pET44a (Novagen, catalog number: 71122-3) was used to construct the expression vector.

Specifically, using the pGEM-TNFRI171 plasmid prepared above as a template, the Met-TNFRI105 gene (SEQ ID NO: 100) was obtained by PCR. This gene was designed with restriction enzyme sites NdeI and HindIII at the 5' and 3' ends, respectively, which allowed cloning of the gene into the pET44a vector.

The primers used for this PCR amplification have the following base sequences:

Forward primer: 5'-acatatggatagcgtgtgcccgc-3'

Reverse primer: 5'-taagctttattaattaaaacactggaac-3'

PCR was initiated with denaturation at 95°C for 5 minutes followed by 25 cycles of denaturation at 95°C for 1 minute, annealing at 60°C for 40 seconds and extension at 72°C for 1 minute, followed by a final extension at 72°C for 10 minutes. The PCR product thus obtained and the pET44a vector were respectively degraded with restriction enzymes (NdeI, HindIII) at 37°C for 3 hours. After enzymatic degradation, the degraded product was electrophoresed on 1% agarose gel, and the DNA band detected at the appropriate size was excised with a razor blade, and DNA extraction kit (GeneAll, catalog number: 102-102) was used. extract. Ligation buffer (2x buffer) was mixed with 50 ng linearized pET44a vector, 200 ng Met-TNFRI105 gene, 1 μL ligase (NEB, catalog number: M2200S) and sterile distilled water to make a total volume of 20 μL, followed by incubation at room temperature for 10 minutes. 2 μl of the reaction solution was taken out and added to XL1-blue competent cells, which were then transformed by heat shock at 37° C. for 2 minutes, followed by static culture in LB solid medium containing ampicillin to obtain colonies. The colony was cultured in an LB liquid medium containing ampicillin, from which a plasmid was isolated and the gene sequence was confirmed. The resulting recombinant plasmid was named pET44a-Met-TNFRI105 (Fig. 1).

Using the pGEM-TNFRI171 plasmid as a template, the Met-TNFRI126 gene (SEQ ID NO: 101) was obtained by PCR. This gene was designed with restriction enzyme sites NdeI and BamHI at the 5' and 3' ends, respectively, which allowed cloning of the gene into the pET44a vector.

The primers used for this PCR amplification have the following base sequences:

Forward primer: 5'-acatatggatagcgtgtgcccgc-3'

Reverse primer: 5'-cggatccttaacaaactgtattctgcttc-3'

The PCR reaction was performed in the same manner as for the Met-TNFRI105 gene. In addition, the same procedure as when constructing the Met-TNFRI105 expression vector was repeated except that restriction enzymes NdeI and BamHI were used. The resulting recombinant plasmid prepared from the culture of the colony grown on the plate was named pET44a-Met-TNFRI126. Cloning of the gene of interest was confirmed by isolating plasmids from colonies and performing base sequencing (Fig. 1).

Using the pGEM-TNFRI171 plasmid as a template, the Met-TNFRI171 gene (SEQ ID NO: 102) was obtained by PCR. This gene was designed with restriction enzyme sites NdeI and BamHI at the 5' and 3' ends, respectively, which allowed cloning of the gene into the pET44a vector.

The primers used for this PCR amplification have the following base sequences:

Forward primer: 5'-acatatggatagcgtgtgcccgc-3'

Reverse primer: 5'-cggatccttatgtggtgcctgagtcctc-3'

The PCR reaction and construction of the E. coli expression vector were performed in the same manner as for the Met-TNFRI126 gene. The resulting recombinant plasmid prepared from the culture of the colony was named pET44a-Met-TNFRI171. Cloning of the gene of interest was confirmed by isolating a plasmid from the colony and performing base sequencing on the plasmid (Fig. 1).

[Preparation Example 2] Construction of TNFRI fragment mutants with improved TNF-α binding ability

(1) Design of TNFRI mutants

Amino acid modifications applied to TNFRI105, TNFRI126 and TNFRI171 are listed in Table 1 below. In the amino acid sequence of SEQ ID NO: 1 of wild-type human TNFRI, the amino acid residues predicted to be involved in binding TNF-α were analyzed. The identified amino acid residues were substituted with other amino acids expected to improve the affinity of TNFRI for TNF-[alpha].

Table 1: List of designed TNFRI amino acid modifications

Mutants produced by introducing the amino acid modifications indicated by numbers 1 to 30 in Table 1 into TNFRI105, TNFRI126, and TNFRI171 were designated as TNFRI105-A1 to TNFRI105-A30, TNFRI126-A1 to TNFRI126-A30, and TNFRI171-A1 to TNFRI171- A30 (symbol "A" represents a candidate mutant with increased affinity. For example, TNFRI105-A1 means TNFRI105 candidate mutant number 1 expected to have increased affinity for TNF-α).

(2) Construction of DNA encoding TNFRI mutants

Site-specific TNFRI mutants were constructed using site-directed mutagenesis. The primers used to construct TNFRI mutants containing the 30 amino acid mutations listed in Table 1 are summarized in Table 2 below.

Specifically, each pair of primers was dissolved in distilled water at a concentration of 20 pM, and used to generate TNFRI plasmids (previously constructed pET44a-Met-TNFRI105, pET44a-Met-TNFRI126, or pET44a-Met) in the presence of Pfu polymerase. -TNFRI171) served as a template for the construction of site-directed mutants by PCR.

Using primers corresponding to A30 and A1 in Table 2, using the pET44a-Met-TNFRI105, pET44a-Met-TNFRI126 or pET44a-Met-TNFRI171 plasmid prepared above as a template, the TNFRI105-A1, TNFRI-126-A1, TNFRI -171-A1, TNFRI105-A30, TNFRI-126-A30 and TNFRI-171-A30 genes were amplified and inserted into the plasmids to construct recombinant plasmids (named pET-TNFRI105_A1, pET-TNFRI-126_A1, pET-TNFRI-126_A1, pET- TNFRI-171_A1, pET-TNFRI105_A30, pET-TNFRI126_A30 and pET-TNFRI171_A30). As described in Table 2 below, subsequently, the plasmids were used as templates to construct pET-TNFRI_A2 to pET-TNFRI_A29.

The composition used for the amplification was as follows: a total of 50.0 µl of a reaction solution containing 1.0 µl of each template plasmid DNA, 1.0 µl of 20 pmole N-primer, 1.0 µl of 20 pmole C-primer, 25.0 µl of 2XPrimeSTAR PCR buffer, 4.0 µl of 200 µM dNTP, 0.5 µl of PrimeSTAR HS DNA polymerase (Takara, catalog number: R044A) and 17.5 μl of distilled water.

PCR was initiated with denaturation at 98°C for 5 minutes followed by 17 cycles of denaturation at 98°C for 30 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 9 minutes, followed by a final extension at 72°C for 10 minutes.

The reaction mixture was treated with DpnI enzyme at 37°C for 2 hours to degrade E. coli-derived DNA while leaving the PCR product intact. 2 μl of the DNA reaction solution was taken out and added to XL1-blue competent cells, which were then transformed by heat shock at 42° C. for 2 minutes, followed by static culture in LB solid medium containing ampicillin to obtain colonies. The colony was cultured in an LB liquid medium containing ampicillin, from which a plasmid was isolated and construction of a site-specific TNFRI mutant was confirmed by base sequencing.

Table 2 Primers used for site-directed mutagenesis

(3) Production of biologically active Met-TNFRI and Met-TNFRI mutants in Escherichia coli

(A) Expression of Met-TNFRI and Met-TNFRI mutants

1 μL of the constructed plasmid was used to transform BL21STAR(DE3) (Invitrogen, catalog number: C6010-03) competent cells by heat shock at 42°C for 1 minute, and incubated on LB plates to form colonies. Escherichia coli BL21STAR (DE3) carrying the expression vector was inoculated into 50 mL of LYP culture solution containing 100 μg/mL ampicillin (yeast extract: MERCK, catalog number: 103753, peptone: BD, catalog number: 243620, NACL: MERCK, catalog number: 1064049025) and incubated for 16 hours at 37°C under aeration. Inoculate the culture into 250 mL of YP broth containing 100 μg/mL ampicillin in a 1 L flask to an O.D. of 0.1 at 600 nm. When the cells were incubated at 37°C until the O.D. at 600 nm was 3-4, IPTG was added at a final concentration of 1.0 nM to induce expression. After IPTG induction, cells were incubated for an additional 3 hours at 37°C under aeration and harvested by centrifugation at 6000 rpm for 20 minutes.

(B) Recovery of insoluble Met-TNFRI and Met-TNFRI mutants

The collected cells were resuspended in a resuspension solution (50 mMTRIS, 0.5 mM EDTA, pH 8.5) and disrupted with a sonicator (Sonics, catalog number: VCX750). After centrifugation at 8000xg and 10°C for 30 minutes, the supernatant was discarded and the pellet was suspended in Wash Buffer 1 (50 mM TRIS, 10 nM EDTA, 0.5% TritonX-100, pH 8.0) and centrifuged at 8000xg and 10°C for 20 minutes. The supernatant was discarded and the resulting pellet was resuspended in the resuspension solution and centrifuged at 8000 xg and 10°C for 20 minutes. The pellet was used immediately or frozen at -80°C for later use.

(C) Solubilization and refolding of Met-TNFRI and Met-TNFRI mutants

The pellet was solubilized in 6 mL of denaturing solution (6-8 M urea or 6-8 M guanidine-HCL, 10 M M dithiothreitol (DTT), 2.0 mM EDTA, 0.2 M NaCl). The precipitated insoluble fraction was filtered off through a 0.45 μm syringe filter. The precipitate solubilized solution was diluted 20-fold in refolding solution (50 mMTRIS, 1.0 mM EDTA, 0.5 ML-arginine, 6.0 mMGSH, 4.0 mMGSSG, 240 mM NaCl, 10 mM KCL, pH 9.0) and stirred gently at 4 °C for 12- 24 hours to induce refolding.

(D) Purification of refolded Met-TNFRI and Met-TNFRI mutants

To purify the refolded Met-TNFRI and Met-TNFRI mutants, the refolding solution was concentrated 20-fold using 3kDAmiconUltra (Millipore, catalog number: UFC900324), followed by XK25/70 column (GE) packed with Superdex75 preparative grade resin (GE). , catalog number: 19-0146-01) were subjected to gel filtration chromatography.

The column was equilibrated with 4-5 volumes of equilibration buffer (50 mM sodium phosphate, 100 mM NaCl, pH 7.0) before loading the refolded sample on the column. After loading 2 mL of sample onto the equilibrated column, the equilibrated solution was passed through the column at a flow rate of 5 mL/min and 5 mL fractions were taken. Only fractions observed to have a purity of 90% or greater (as analyzed by SDS-PAGE) were used. By this method, Met-TNFRI105, Met-TNFRI126, Met-TNFRI171, Met-TNFRI105 mutant, Met-TNFRI126 mutant and Met-TNFRI171 mutant were purified (Fig. 2).

[Experimental Example 1] Analysis of the affinity of Met-TNFRI to ligand (TNF-A)

Met-TNFRI and Met-TNFRI mutants (both with a purity of 90% or higher) were quantitatively analyzed using the Bradford method and affinity for TNF-α was determined using ELISA.

TNFRI190 (consisting of 190 amino acid residues extending from position 22 to position 211 in the amino acid sequence of TNFRI of SEQ ID NO: 1; R&D, catalog number: 636-R1-025-CF) was prepared in 100 mL at a concentration of 1 μg/mL The amount was plated into a 96-well plate and fixed at 4°C for 16 hours. Each well was washed 3 times with 300 μL wash buffer (0.05% Tween-20, PBS, pH 7.4) and incubated with 300 μL blocking buffer (5% skim milk, PBS, pH 7.4) for 2 hours at room temperature. Subsequently, each well was washed as mentioned above. Samples of interest were serially diluted to concentrations of 500 nM, 125 nM, 31 nM, 7.8 nM, 1.9 nM, 0.48 nM, 0.12 nM and 0.03 nM and added in 100 μL to each well in duplicate. 100 μL of 50 ng/mL TNF-α was added to each well, followed by incubation at room temperature for 2 hours. After the wells were washed with washing buffer, 100 μg/mL TNF-α antibody solution was diluted 1/1000, added in an amount of 100 μL per well, and incubated at room temperature for 2 hours. After the plate was washed with the washing buffer, the substrate was added in an amount of 100 μL per well and reacted at room temperature for 15 minutes. In each well, 100 μL of the substrate 3,3',5,5'-tetramethylbenzidine (RnD, catalog number: DY999) was reacted at room temperature for 15 minutes, and then 50 μL of 1.0 M sulfuric acid (Samchun Chemical , catalog number: S2129) to terminate the reaction. Absorbance at 450 nm and 540 nm was read on a Vmax reader (MD, model: VersaMax).

The affinity of Met-TNFRI and Met-TNFRI mutants for TNF-α was determined by measuring the change in absorbance with concentration and evaluating the _IC50 value by measuring the change in absorbance with concentration. From this the affinity of the Met-TNFRI mutants relative to wild-type TNFRI was determined. The results are shown in Table 3 below. Data for representative mutants TNFRI126-A30 and TNFRI171-A30 are shown in FIG. 3 . It can be seen from Fig. 3 that TNFRI126 and TNFRI171 basically have the same effect. Furthermore, the TNFRI mutants of the present invention showed up to 1,400% improvement in affinity compared to wild-type TNFRI.

[Experimental Example 2] Assay for evaluating the biological activity of Met-TNFRI and Met-TNFRI mutants by neutralizing the cytotoxicity of TNF-α

Purified Met-TNFRI and Met-TNFRI mutants were quantitatively analyzed using the Bradford method and applied to WEHI cells (ATCC, catalog number: CRL-2148) susceptible to a certain concentration of TNF-α so that The protection of these cells from the cytotoxic effects of TNF-[alpha] was measured.

Specifically, WEHI (ATCC, catalog number: CRL-2148) cells were suspended at a density of ² ×10 cells in RPMI medium supplemented with 5% fetal bovine serum, 50 U/mL penicillin, and 50 mg/mL streptomycin . The cell suspension was inoculated into a 96-well microtiter plate at an amount of 100 μL/well and cultured at 37° C. under a 5% CO ₂ atmosphere for 24 hours. 100 μL of 70 pg/mL TNF-α in medium containing actinomycin D was added to each well. 100 μg/mL of each mutant sample was diluted from 3.0 nM to 0.18 pM in PBS and added to each well containing TNF-α. As a control, ENBREL (AMGEN, chemical name: etanercept) was applied at a concentration of 0.04 pM to 3.0 nM, while wild-type TNFRI was used at a concentration of 12 pM to 200 nM. WEHI cells were incubated at 37°C under 5% CO ₂ atmosphere for 24 hours and then incubated with MTT reagent (MTT assay kit, Roche, catalog number: 11455007001 ) for an additional 4-6 hours. Add solubilization reagent to each well. Dissolution of purple formazan was identified after 24 hours of incubation, after which the absorbance at 570 nm was read on a Vmax reader. The activity of Met-TNFRI and Met-TNFRI mutants was determined by measuring _ND50 values by means of absorbance as a function of concentration and calculating relative activity relative to wild-type TNFRI. The results are summarized in Table 4 below. Representative mutants Data for representative mutants TNFRI126-A30, TNFRI171-A2, TNFRI171-A9, TNFRI171-A21, TNFRI171-A22 and TNFRI171-A30 are shown in FIG.

As can be seen from FIG. 4A , it was observed that TNFRI126 and TNFRI171 exhibit essentially the same effect. In addition, the biological activity of the Met-TNFRI mutant of the present invention is higher than that of wild-type TNFRI. For example, TNFRI171-A2, TNFRI171-A21 and TNFRI171-A22 were 200-fold more biologically active than wild-type TNFRI as shown by the assay for neutralization of cytotoxicity of TNF-α (Table 4). Thus, the cytotoxic neutralizing activity of TNFRI mutants increases with increasing affinity for TNF-α.

[Experimental Example 3] Met-TNFRI and resistance of Met-TNFRI to protease

Analysis of protease resistance of Met-TNFRI and Met-TNFRI mutants. First, the total protein concentration of purified protein (eg, TNFRI) is determined using the Bradford method. The purified protein was degraded by treatment with porcine trypsin (SIGMA, catalog number: P7545) in an amount of 40% of the total protein concentration. The half-life of each mutant was determined by trypsinology and compared with that of wild-type TNFRI.

Specifically, the concentrations of MET-TNFRI and each mutant were determined by the Bradford method and adjusted to 100 μg/mL using PBS. Each protein sample thus obtained was placed in an amount of 250 µL in a 500 µL-centrifuge tube. 30 μL of 0.1 M sodium phosphate containing 10 μg of trypsin was added to the tube, followed by incubation at 37°C. At 0 min, 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 60 min after the incubation, take 30 µL of each sample and mix with 270 µL of a solution containing 5 µL of protease inhibitors (Roche, catalog number: 11836170001). 5% BSA solutions were mixed and stored in liquid nitrogen. The half-life of each Met-TNFRI mutant was determined by quantitatively analyzing the intact protein using ELISA (RnD, catalog number: DY225). The half-life of each mutant is expressed as a percentage of Met-TNFRI (Tables 3 and 4). As shown, Met-TNFRI mutants with increased affinity were found to be more susceptible to degradation by proteases.

Table 3: Determination of affinity and protease resistance of mutants by ELISA assay

Table 4: Biological activity determined by cytotoxicity assay and protease resistance of mutants

[Preparation Example 3] Selection of mutants with improved protease resistance

(1) Design of TNFRI mutants

Mutants were engineered to be protease resistant. With the help of the peptide cutter program provided by Expasy (Expert Protein Analysis System), by analyzing the cleavage sites of 10 common proteases present in the gastrointestinal tract, cells and blood (http://www.expasy.org/tools/peptidecutter/ ), deduce the mutagenic position in the amino acid sequence of TNFRI117. Amino acid residues in the putative cleavage site were substituted with amino acids that were resistant to proteases and caused as little structural change as possible. The PAM250 matrix was used to identify the amino acids to replace putative residues. Referring to the PAM250 matrix proposing 19 amino acids with positive, zero, and negative values for each amino acid, amino acids that were not cleaved by proteases and had positive or zero values were determined as substitutes.

Table 5: Protease-specific amino acids and resistance-causing amino acids

protease Amino acids recognized and cleaved Amino acids causing cleavage resistance Arg-C protease R H or Q Asp-N endopeptidase -D N or Q Chymotrypsin [FYWML], not before P S or I, H Enterokinase K N or Q glutamyl endopeptidase E. N or Q

Lys-C K N or Q Lys-N K N or Q proline endopeptidase H, K or R, -P A or S thrombin R N or Q Trypsin K N or Q

The designed TNFRI mutants are listed in Table 6 below.

List of TNFRI mutants designed in Table 6

mutant number mutation mutant number mutation TNFRIx-R1 K48Q TNFRIx-R47 M109I TNFRIx-R2 K48N TNFRIx-R48 M109V TNFRIx-R3 Y49I TNFRIx-R49 E113Q TNFRIx-R4 Y49H TNFRIx-R50 E113N TNFRIx-R5 P52A TNFRIx-R51 D120N TNFRIx-R6 P52S TNFRIx-R52 D120Q TNFRIx-R7 K61Q TNFRIx-R53 R121H TNFRIx-R8 K61N TNFRIx-R54 R121Q TNFRIx-R9 K64Q TNFRIx-R55 D122N TNFRIx-R10 K64N TNFRIx-R56 D122Q TNFRIx-R11 Y67I TNFRIx-R57 R128H TNFRIx-R12 Y67H TNFRIx-R58 R128Q TNFRIx-R13 L68I TNFRIx-R59 K129Q TNFRIx-R14 L68V TNFRIx-R60 K129N TNFRIx-R15 Y69I TNFRIx-R61 Y132I TNFRIx-R16 Y69H TNFRIx-R62 Y132H TNFRIx-R17 D71N TNFRIx-R63 R133H TNFRIx-R18 D71Q TNFRIx-R64 R133Q TNFRIx-R19 D78N TNFRIx-R65 Y135I

mutant number mutation mutant number mutation TNFRIx-R20 D78Q TNFRIx-R66 Y135H TNFRIx-R21 D80N TNFRIx-R67 W136H TNFRIx-R22 D80Q TNFRIx-R68 W136S TNFRIx-R23 R82H TNFRIx-R69 E138Q TNFRIx-R24 R82Q TNFRIx-R70 E138N TNFRIx-R25 E83Q TNFRIx-R71 L140I TNFRIx-R26 E83N TNFRIx-R72 L140V TNFRIx-R27 E85Q TNFRIx-R73 F141I TNFRIx-R28 E85N TNFRIx-R74 F141V TNFRIx-R29 F89I TNFRIx-R75 F144I TNFRIx-R30 F89V TNFRIx-R76 F144V TNFRIx-R31 E93Q TNFRIx-R77 L150I TNFRIx-R32 E93N TNFRIx-R78 L150V

(* In the following examples, the symbol x represents 108, 126 or 171 for mutant numbers R1 to R76; 126 or 171 for mutant numbers R77 to R84; and 171 for mutant numbers R85 to R92. The symbol "R " represents a candidate mutant with increased protease resistance. For example, TNFRIx-R1 means a candidate mutant of TNFRIx expected to have increased protease resistance 1).

(2) Construction of TNFRI108 gene and expression vector

In order to express the soluble TNFRI108 protein in Escherichia coli, the commercially available vector pET44a (Novagen, catalog number: 71122-3) was used to construct the expression vector.

Specifically, the TNFRI108 gene was obtained by PCR using the pGEM-TNFRI171 plasmid constructed in Preparation Example 1 as a template in the presence of the following primers. This gene was designed with restriction enzyme sites SmaI and HindIII at the 5' and 3' ends, respectively, which allowed cloning of the gene into the pET44a vector.

Forward primer: 5'-ccccggggcgatgacgatgacaaagatagcgtgtgcccg-3'

Reverse primer: 5'-taagcttattacagggagcaattaaaacactgg-3'

PCR was performed as in (2) of [Preparation Example 1]. According to the manufacturer's instructions, 2 μl of the PCR product was inserted into the pcDNA3.3TOPOTA vector (Invitrogen, catalog number: K8300-01). 2 μl of the reaction solution was removed and added to XL1-blue competent cells (RBC, catalog number: RH119-J80), which were then transformed by heat shock at 42 °C for 1 minute, followed by LB solid medium containing ampicillin cultured statically to obtain colonies. The colonies were cultured in LB liquid medium containing ampicillin, from which plasmids were isolated and gene sequences were confirmed with fluorescent ddNTPs (Solgent, Korea). The resulting recombinant plasmid was named pcDNA3.3-TNFRI108.

The pcDNA3.3-TNFRI108 and pET44a prepared above were each treated with restriction enzymes (SmaI, HindIII) at 37°C for 3 hours. After enzymatic degradation, the degraded product was electrophoresed on 1% agarose gel, and the DNA band detected at the appropriate size was excised with a razor blade, and DNA extraction kit (GeneAll, catalog number: 102-102) was used. extract. The pET44a vector was ligated with the TNFRI108 gene in the presence of ligase (NEB, catalog number: M2200S). 2 μl of the reaction solution was removed and added to XL1-blue competent cells (RBC, catalog number: RH119-J80), which were then transformed by heat shock at 37°C for 2 minutes, followed by LB solid medium containing ampicillin cultured statically to obtain colonies. The colony was cultured in LB liquid medium containing ampicillin, from which a plasmid was isolated to construct a TNFRI108 expression vector (Fig. 5). The resulting recombinant expression vector was named pET44a-NusA-TNFRI108.

(3) Construction of DNA encoding TNFRI108 single mutant

Construction of TNFRI108 single mutants using site-directed mutagenesis. The primers used to construct TNFRI single mutants are summarized in Table 7 below.

A DNA encoding a TNFRI108 single mutant was constructed in the same manner as in (2) of [Preparation Example 2], but using pET44a in the presence of each primer pair for mutants R1 to R76 in Table 7 -NusA-TNFRI108 plasmid as template.

Table 7 Primers for site-directed mutagenesis

(4) Expression of TNFRI108 and TNFRI108 mutants

Heat-shocked at 42°C for 1 minute, BL21Star(DE3) (Invitrogen, catalog number: C6010-03) competent cells were transformed with 1 μl of the constructed plasmid and incubated on LB plates to form colonies. Escherichia coli BL21Star (DE3) carrying the pET44a-NusA-TNFRI108 and TNFRI108 mutant vectors was inoculated into 5 mL of LB medium (BD, catalog number: 244620) containing 100 μg/ml ampicillin and incubated at 37° C. under aeration for 16 hours . The culture was inoculated into 50 mL medium containing 100 μg/ml ampicillin and grown to 0.6-0.8 O.D. IPTG (Sigma, catalog number: I9003) was added at a final concentration of 1.0 mM to induce expression. After IPTG induction, cells were incubated for an additional 3 hours at 37°C under aeration and harvested by centrifugation at 5000 rpm for 20 minutes.

(5) Purification of TNFRI108 and TNFRI108 mutants

After disrupting the collected cells, the supernatant is purified into the target protein with a purity of 90% or higher using metal affinity chromatography followed by hydrophobic chromatography.

Harvested cells were resuspended in solution (50 mM Tris (pH 8.5)) and disrupted with a sonicator (Sonics, catalog number: VCX750). After centrifugation at 12,000 rpm for 20 minutes, the supernatant was recovered. Hypercell resin (Pall, catalog number: 20093-010) was loaded into a 96-well filter plate (Pall, catalog number: PN5065) at an amount of 300 μL/well, washed with 4 column volumes of distilled water and washed with 2 column volumes of _0.1MNiCl2 treatment to bind the nickel ions to the resin. They were washed with two column volumes of distilled water and then equilibrated with 6 column volumes of equilibration buffer (25 mM Tris, 0.1 M NaCl (pH 8.5)). 2 column volumes of sample were added to the resin and unbound sample was removed by flowing through 4 column volumes of equilibration buffer. After washing with 10 column volumes of wash buffer (25 mM Tris, 0.1 M NaCl, 50 mM imidazole (pH 8.5)), wash with two column volumes of eluent (25 mM Tris, 0.1 M NaCl, 250 mM imidazole (pH 8.5)). TNFRI108 bound to the column was detached.

1.0 M ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) was diluted to 400 mM in the protein solution eluted from the metal affinity chromatography. Phenyl Sepharose resin (GE, catalog number: 17-108201) was loaded into a 96-well filter plate at an amount of 300 μL/well and equilibrated with 6 column volumes of equilibration buffer (20 mM sodium phosphate (Na ₂ PO ₄ ), 400mM ammonium sulfate (pH7.0)) balance. The eluate from metal affinity chromatography was added to the column and washed with 2 column volumes of equilibration buffer to remove unbound protein. After washing with 10 column volumes of wash buffer (20 mM sodium phosphate (Na ₂ PO ₄ ), 160 mM ammonium sulfate (pH 7.0)), wash with 6 column volumes of eluent (20 mM sodium phosphate (pH 7.0) ) for elution. The eluate was concentrated to at least 100 μg/ml before quantitative analysis using the Bradford method. The purity of all purified samples was assessed by SDS-PAGE (Figure 6).

[Experimental Example 4] Protease Resistance of TNFRI108 and TNFRI108 Mutants

Protease resistance of NFRI108 and TNFRI108 mutants was determined. First, the total concentration of purified protein (eg, TNFRI108) is determined using the Bradford method. The purified protein was digested using porcine trypsin in an amount of 40% of the total protein concentration. Based on trypsin treatment, the half-life of each mutant was determined and compared with the half-life of the TNFRI108 polypeptide fragment.

To evaluate the resistance of the TNFRI108 mutants to proteases, the half-lives of the mutants were measured after treatment with trypsin (Sigma, catalog number: P7545) and compared with those of TNFRI108. Resistance to trypsin of representative mutants TNFRI108-R14, TNFRI108-R64 and TNFRI108-R68 is shown in FIG. 7 . The same method as in [Experimental Example 3] was repeated except that 250 µl of each protein sample was treated with 6 µg of trypsin. The results are summarized in Table 8 below.

Table 8: Protease resistance of TNFRI108 mutants (reacted at 37°C)

mutant number Resistance compared to TNFRI108 mutant number Resistance compared to TNFRI108 TNFRI108-R1 73% TNFRI108-R39 89% TNFRI108-R2 48% TNFRI108-R40 90% TNFRI108-R3 5% TNFRI108-R41 125% TNFRI108-R4 6% TNFRI108-R42 113% TNFRI108-R5 26% TNFRI108-R43 110% TNFRI108-R6 57% TNFRI108-R44 96%32 --> TNFRI108-R7 108% TNFRI108-R45 37% TNFRI108-R8 111% TNFRI108-R46 57% TNFRI108-R9 43% TNFRI108-R47 158% TNFRI108-R10 14% TNFRI108-R48 103% TNFRI108-R11 twenty four% TNFRI108-R49 51% TNFRI108-R12 30% TNFRI108-R50 twenty three% TNFRI108-R13 166% TNFRI108-R51 67% TNFRI108-R14 168% TNFRI108-R52 71%

mutant number Resistance compared to TNFRI108 mutant number Resistance compared to TNFRI108 TNFRI108-R15 14% TNFRI108-R53 116% TNFRI108-R16 13% TNFRI108-R54 119% TNFRI108-R17 17% TNFRI108-R55 83% TNFRI108-R18 81% TNFRI108-R56 28% TNFRI108-R19 89% TNFRI108-R57 141% TNFRI108-R20 106% TNFRI108-R58 116% TNFRI108-R21 ND TNFRI108-R59 86% TNFRI108-R22 ND TNFRI108-R60 84% TNFRI108-R23 ND TNFRI108-R61 96% TNFRI108-R24 ND TNFRI108-R62 89% TNFRI108-R25 75% TNFRI108-R63 91% TNFRI108-R26 75% TNFRI108-R64 213% TNFRI108-R27 93% TNFRI108-R65 171% TNFRI108-R28 149% TNFRI108-R66 111% TNFRI108-R29 ND TNFRI108-R67 109% TNFRI108-R30 ND TNFRI108-R68 288% TNFRI108-R31 70% TNFRI108-R69 107% TNFRI108-R32 50% TNFRI108-R70 105% TNFRI108-R33 70% TNFRI108-R71 74% TNFRI108-R34 55% TNFRI108-R72 103%

(ND; not detected)

(6) Construction of expression vectors carrying Met-TNFRI126 and Met-TNFRI126 mutants

The protease resistance of the single mutant demonstrated in TNFRI108 was also established in Met-TNFRI126 and Met-TNFRI126 mutants. In this case, TNFRI108-R7, TNFRI108-R8, TNFRI108-R9, TNFRI108-R10, TNFRI108-R14, TNFRI108-R64, TNFRI108-R65, TNFRI108-R68, TNFRI108-R73 and TNFRI108-R74 were replaced with Met-TNFRI126 to test their resistance to proteases.

Plasmids carrying Met-TNFRI126 and Met-TNFRI126 mutants were constructed in the same manner as in (2) of [Preparation Example 2], but in Table 7 with mutants R7, R8, R9, R10, R14, R64 In the presence of each primer pair corresponding to R65, R68, R73, and R74, the pET44a-Met-TNFRI126 plasmid constructed in [Preparation Example 1] was used as a template.

(7) Production and purification of Met-TNFRI126 and Met-TNFRI126 mutants

Met-TNFRI126 and Met-TNFRI126 mutants were prepared and purified in the same manner as in (3) of [Preparation Example 2], but using 1 µl of each plasmid constructed above.

[Experimental Example 5] Resistance of Met-TNFRI126 and Met-TNFRI126 Mutants to Proteases

Protease resistance was measured in the same manner as in [Experimental Example 4], but using Met-TNFRI126 and a Met-TNFRI126 mutant instead of TNFRI108 (Table 9). High protease resistance was also detected in R14, R64, R68, R73 and R74 which were observed to have high protease resistance in TNFRI108, while R9 and R10 which showed low protease resistance in TNFR108 were also measured to have low resistance .

Table 9: Protease resistance relative to Met-TNFRI126

mutant number Resistance relative to Met-TNFRI126 (%) TNFRI126-R7 87% TNFRI126-R8 95% TNFRI126-R9 65% TNFRI126-R10 30% TNFRI126-R14 143% TNFRI126-R64 152% TNFRI126-R65 99% TNFRI126-R68 120% TNFRI126-R73 159%

mutant number Resistance relative to Met-TNFRI126 (%) TNFRI126-R74 169%

(8) Construction of expression vectors carrying Met-TNFRI171 and Met-TNFRI171 mutants

Single mutants resulting from mutations in the fourth domain of TNFRI were selected from the designed single mutants listed in Table 6 of (1) of [Preparation Example 3], and were prepared as Met-TNFRI171.

For this purpose, expression vectors for the expression of Met-TNFRI171 and Met-TNFRI171 mutants in E. coli were constructed. Using the primers for the mutants shown in Table 10 below, the gene encoding the Met-TNFRI171 mutant was constructed by PCR with the pET44a-Met-TNFRI171 plasmid serving as a template.

Plasmids carrying Met-TNFRI171 and Met-TNFRI171 mutants were constructed in the same manner as in (2) of [Preparation Example 2], but in the presence of each primer pair shown in Table 10, using [ The pET44a-Met-TNFRI171 plasmid constructed in Preparation Example 1] was used as a template.

Table 10: Primers for site-directed mutagenesis

(9) Production and purification of Met-TNFRI171 and Met-TNFRI171 mutants

Met-TNFRI171 and Met-TNFRI1716 mutants were prepared and purified in the same manner as in (3) of [Preparation Example 2], but using 1 µl of each plasmid constructed above.

[Experimental Example 6] Protease Resistance of Met-TNFRI171 and Met-TNFRI171 Mutants

Protease resistance was determined in the same manner as in [Experimental Example 3], but using Met-TNFRI171 and Met-TNFRI171 mutation instead of TNFRI108 (Table 11). Resistance to trypsin of representative mutants Met-TNFRI171-R83, Met-TNFRI171-R84 and Met-TNFRI171-R92 is shown in FIG. 8 .

Table 11: Protease resistance relative to Met-TNFRI171

mutant number Resistance relative to Met-TNFRI171 TNFRI171-R77 120% TNFRI171-R78 87% TNFRI171-R79 113%

mutant number Resistance relative to Met-TNFRI171 TNFRI171-R80 103% TNFRI171-R81 57% TNFRI171-R82 68% TNFRI171-R83 187% TNFRI171-R84 187% TNFRI171-R85 116% TNFRI171-R86 90% TNFRI171-R87 111% TNFRI171-R88 99% TNFRI171-R89 114% TNFRI171-R90 102% TNFRI171-R91 117% TNFRI171-R92 127%

[Example 1] Construction of mutant (Superlead) with improved affinity to TNF-α and improved protease resistance

(1) Superlead design

As assessed in [Preparation Example 2], the mutant with improved affinity for TNF-α was highly susceptible to proteases. In order to impart improved protease resistance to these mutants, the mutation for improving protease resistance confirmed in [Preparation Example 3] was combined with the mutation for improving affinity confirmed in [Preparation Example 2] . As a result, Superlead mutants were constructed according to the length of TNFRI, as shown in Table 12 to Table 14.

Table 12: List of amino acid modifications designed in TNFRI171

Table 13: List of amino acid modifications designed in TNFRI126

Table 14: List of amino acid modifications designed in TNFRI105

The amino acid sequences of TNFRI171, TNFRI126 and TNFRI105 mutants to which combined mutations are applied are shown in SEQ ID NOS: 6 to 99.

(2) Construction of DNA encoding TNFRI mutants

Site-specific TNFRI mutants were constructed using site-directed mutagenesis. The primers used to construct the TNFRI mutants comprising the amino acid mutations of Table 12 to Table 14 are summarized in Table 15 below. A DNA encoding a TNFRI mutant was constructed in the same manner as in (2) of [Preparation Example 2], but using the primer pairs shown in Table 15.

Table 15: Primers for site-directed mutagenesis

(3) Production of biologically active Met-TNFRI and Met-TNFRI mutants in Escherichia coli

Met-TNFRI and Met-TNFRI mutants constructed above were produced and purified according to the method described in (3) of [Preparation Example 2].

[Experimental Example 7] Determination of the affinity of Met-TNFRI mutants to TNF-α

The affinity of Met-TNFRI to TNF-α was determined using the same method as in [Experimental Example 1] (Tables 16 to 18).

[Experimental Example 8] Resistance of Met-TNFRI and Met-TNFRI Mutants to Proteases

Protease resistance was measured in the same manner as in [Experimental Example 3] (Table 16 to Table 18). Resistance to trypsin of representative Superlead mutants TNFRI171-S31 , TNFRI171-S47, TNFRI171-S53 and TNFRI171-S63 is shown in FIG. 9 . The TNFRI171 fragment was used as a control.

Table 16: Affinity and protease resistance of Superlead mutants

mutant number Relative affinity for ligand (%) Relative protease resistance (%) wild type (TNFRI171) 100 100 TNFRI171-S31 563 97 TNFRI171-S32 668 48 TNFRI171-S33 568 50

mutant number Relative affinity for ligand (%) Relative protease resistance (%) TNFRI171-S34 700 48 TNFRI171-S35 521 44 TNFRI171-S36 510 120 TNFRI171-S37 668 57 TNFRI171-S38 589 61 TNFRI171-S39 705 57 TNFRI171-S40 773 56 TNFRI171-S41 558 12642 --> TNFRI171-S42 416 67 TNFRI171-S43 579 68 TNFRI171-S44 694 69 TNFRI171-S45 679 67 TNFRI171-S46 805 81 TNFRI171-S47 758 98 TNFRI171-S48 594 36 TNFRI171-S49 547 14 TNFRI171-S50 589 14 TNFRI171-S51 537 14 TNFRI171-S52 626 15 TNFRI171-S53 1017 123 TNFRI171-S54 900 125 TNFRI171-S55 973 85 TNFRI171-S56 510 129 TNFRI171-S57 773 159 TNFRI171-S58 978 123 TNFRI171-S59 684 148 TNFRI171-S60 775 132 TNFRI171-S61 778 112 TNFRI171-S62 1196 105 TNFRI171-S63 1354 124

Table 17: Affinity and protease resistance of Superlead mutants

mutant number Relative affinity for ligand (%) Relative protease resistance (%) wild type (TNFRI126) 100 100 TNFRI126-S31 457 137 TNFRI126-S36 489 159 TNFRI126-S41 432 157 TNFRI126-S46 412 87 TNFRI126-S47 501 94 TNFRI126-S48 477 84 TNFRI126-S53 522 156 TNFRI126-S54 448 161 TNFRI126-S56 602 172 TNFRI126-S57 465 191 TNFRI126-S59 463 171 TNFRI126-S60 397 163 TNFRI126-S62 543 12943 --> TNFRI126-S63 486 142

Table 18: Affinity and protease resistance of Superlead mutants

mutant number Relative affinity for ligand (%) Relative protease resistance (%) wild type (TNFRI105) 100 100 TNFRI105-S31 453 153 TNFRI105-S36 459 167 TNFRI105-S41 403 182 TNFRI105-S46 547 137 TNFRI105-S47 497 143

[Experimental Example 9] The therapeutic efficacy of 8 kinds of TNFRI171 mutants (S54, S62, A2, A9, S36, S57, S58, S63) on carrageenan-induced mouse paw edema

The in vivo therapeutic efficacy of these mutants was determined by this experiment. The therapeutic efficacy of the mutants of the invention was determined in vivo. For this purpose, eight mutants exhibiting high protease resistance and/or TNF-α affinity were injected subcutaneously into mice with carrageenan-induced paw edema, and the size of paw edema and paw medial IL-6 levels.

Eight kinds of TNFRI171 mutants (TNFRI171-S54, TNFRI171-S62, TNFRI171-A2, TNFRI171-A9, TNFRI171-S36, TNFRI171-S57, TNFRI171-S58, and TNFRI171-S63) were prepared at 0.25 mg/ml, 0.25 mg/ml Wild type (WT, TNFRI171 fragment), vehicle (50 mM NaPO ₄ , 100 mM NaCl, pH 7.0), 0.5 mg/ml Enbrel (Wyeth). 5-week-old ICR mice (20-25 g) were purchased from OrientBio (CRJ) and acclimatized in Gyeonggi Bio Center for about 7 days before use. They were allowed free access to water and food under automated conditions of temperature (23 ± 2 °C), humidity (55 ± 5%), and light/dark cycle (12 h). Each group consisted of 5 mice. As an inducer of inflammation, λ-carrageenan (Sigma) was dissolved in distilled water to form a 1% solution. This carrageenan solution was administered in a 50 μL dose into the right paw by intrapedal injection using a fine needle syringe (BD).

Drugs and substances of interest are administered according to their PK properties. Enbrel, which has a long half-life, was administered 2 hours before carrageenan injection, while the 8 mutants, wild type and vehicle were administered 30 minutes before carrageenan injection. They were administered by subcutaneous injection (SC) into the neck. Enbrel was injected at a dose of 5mg/kg/10mL, compared with 2.5mg/kg/10mL for wild-type and candidate.

The volume of the right paw was measured using a volumetric instrument (UgoBasile) before carrageenan injection and at 1, 2, 3 and 4 hours after injection. A mark was made on the ankle of the right paw prior to the measurement, thus avoiding the variability of each measurement. Four hours after measurement of edema size, all animals were euthanized with _CO2 and the right paw was excised and stored at -70°C in a cryogenic freezer (Thermo) for subsequent analysis. To measure IL-6 levels in paw tissue, paws were thawed and finely minced using scissors and ground in 500 μl of PBS using a homogenizer. After centrifugation at 10,000 rpm for 5 minutes, the supernatant was divided into 100 μL for protein analysis and 200 μL for IL-6 analysis and stored at -70°C in a cryogenic freezer (Thermo). Quantitative analysis of protein was performed using the Bradford method, while IL-6 levels were determined using an ELISA kit (Komabiotech) according to the manufacturer's instructions and normalized to the amount of protein. IL-6 levels are expressed as a percentage based on the vehicle group and are shown in FIG. 10 .

As can be seen from FIG. 10A , 2 hours after the intrapedal injection of carrageenan, except for the S63 group and the Ebrel group, it was observed that the edema in all experimental groups was significantly reduced in size compared with the vehicle group. Four hours after intrapaw injection of carrageenan, the edema in the paws of all test groups had been significantly reduced (p<0.01 ) compared to the vehicle group. As can be seen from FIG. 10B , 4 hours after the intrapedal injection of carrageenan, compared with the vehicle group, the levels of the inflammatory cytokine IL-6 in all test groups were significantly decreased (p<0.01). Inhibitory activity against IL-6 was striking in the S54, S62, S36, S57, S58 and S63 groups compared to the Enbrel group, with the highest activity seen in the S58 group (p<0.01).

[Experimental Example 10] Comparison of the therapeutic efficacy of TNFRI171-S63 and wild type (TNFRI171 fragment) on carrageenan-induced mouse paw edema

Considering the physical properties and in vivo results of TNFRI171 mutants, a mutant (TNFRI171-S63) was selected and compared with vehicle (50mM NaPO ₄ , 100mM NaCl, pH7.0), Enbrel (Wyeth) and wild type (TNFRI171 fragment) . Wild type was formulated at 0.25 mg/ml, while TNFRI171-S63 mutant and Enbrel were each formulated to a concentration of 0.5 mg/ml. The volume of paw edema and IL-6 level were measured in the same manner as in [Experimental Example 9] to evaluate the therapeutic efficacy of the mutant in vivo. The results are shown in Figure 11.

As can be seen from FIG. 11A , 2 hours after intrapedal injection of carrageenan, a significant reduction in size of edema was observed in the group administered with TNFRI171-S63 at a dose of 5 mg/kg compared to the vehicle group. Four hours after intrapedal injection of carrageenan, the edema had been significantly reduced in the Enbrel group and the TNFRI171-S63 group (5 mg/kg) compared to the vehicle group. The edema reduction produced in the NFRI171-S63 group (5 mg/kg) was greater than that of the positive group administered with Enbrel at a dose of 5 mg/kg ( FIG. 11A ).

As can be seen from Figure 11B, 4 hours after intra-foot injection of carrageenan, except for the wild-type group (2.5mg/kg), the levels of inflammatory cytokine IL-6 in all experimental groups were significantly lower than those of the vehicle group ( p<0.01) (FIG. 11B). Inhibitory activity against IL-6 was striking in both groups administered TNFRI171-S63 at doses 2.5 mg/kg and 5 mg/kg compared to the Enbrel group (5 mg/kg). At the same concentration, wild type and Enbrel were found to reduce IL-6 to a similar extent. Therefore, TNFRI171-S63 showed superior edema and IL-6 inhibitory effects compared with wild-type and Enbrel.

Claims (17)

1. A modified tumor necrosis factor receptor-1 polypeptide, whose amino acid residue modification is selected from: Selected from L68V/S92M/H95F/R97P/H98A, L68V/S92H/H95F/R97P/H98A, L68V/S92I/H95F/R97P/H98A/K161Q, L68V/S92I/H95F/R97P/H98A/K161N, L68V/S92M/ H95F/R97P/H98A/K161Q, L68V/S92M/H95F/R97P/H98A/K161N, L68V/S92M/H95F/R97P/H98A/D207N, L68V/S92H/H95F/R97P/H98A/K161Q, L68V/S92F/H95 Amino acid residue modification of R97P/H98A/K161N, L68V/S92H/H95F/R97P/H98A/D207N, L68V/S92I/H95F/R97P/H98G/K161Q and L68V/S92M/H95F/R97P/H98G/K161N, wherein the polypeptide Other amino acids except the modified amino acid are identical to amino acids 41-211 of the wild-type tumor necrosis factor receptor-1 polypeptide shown in SEQ ID NO: 1; Selected from L68V/S92I/H95F/R97P/H98A, L68V/S92M/H95F/R97P/H98A, L68V/S92H/H95F/R97P/H98A, L68V/S92I/H95F/R97P/H98A/K161Q, L68V/S92I/H95F/ R97P/H98A/K161N, L68V/S92M/H95F/R97P/H98A/K161Q, L68V/S92M/H95F/R97P/H98A/K161N, L68V/S92H/H95F/R97P/H98A/K161Q, L68V/S92H/H95F/R97 Amino acid residue modification of H98A/K161N, L68V/S92I/H95F/R97P/H98G/K161Q and L68V/S92M/H95F/R97P/H98G/K161N, wherein the amino acid of the polypeptide except the modified amino acid is the same as shown in SEQ ID NO: 1 Amino acids 41-166 of the wild-type tumor necrosis factor receptor-1 polypeptide are identical; and Selected from L68V/S92I/H95F/R97P/H98A, L68V/S92M/H95F/R97P/H98A, L68V/S92H/H95F/R97P/H98A, L68V/S92I/H95F/R97P/H98G and L68V/S92M/H95F/R97P/ Amino acid residue modification of H98G, wherein the amino acid of the polypeptide except the modified amino acid is the same as amino acid 41-145 of the wild-type tumor necrosis factor receptor-1 polypeptide represented by SEQ ID NO:1. 2. The tumor necrosis factor receptor-1 polypeptide of modification according to claim 1, it is selected from SEQIDNO:44, SEQIDNO:49, SEQIDNO:61, SEQIDNO:62, SEQIDNO:64-72, SEQIDNO:75, SEQIDNO :78 and the amino acid sequences of SEQ ID NO:86-98. 3. A polypeptide complex comprising two or more copies of the modified tumor necrosis factor receptor-1 polypeptide of claim 1 or 2, said copies being covalently linked to each other. 4. A modified tumor necrosis factor receptor-1 polypeptide, the amino acid residue modification of which is selected from: Selected from L68V/S92M/H95F/R97P/H98A, L68V/S92H/H95F/R97P/H98A, L68V/S92I/H95F/R97P/H98A/K161Q, L68V/S92I/H95F/R97P/H98A/K161N, L68V/S92M/ H95F/R97P/H98A/K161Q, L68V/S92M/H95F/R97P/H98A/K161N, L68V/S92M/H95F/R97P/H98A/D207N, L68V/S92H/H95F/R97P/H98A/K161Q, L68V/S92F/H95 Amino acid residue modification of R97P/H98A/K161N, L68V/S92H/H95F/R97P/H98A/D207N, L68V/S92I/H95F/R97P/H98G/K161Q and L68V/S92M/H95F/R97P/H98G/K161N, wherein the polypeptide Other amino acids except the modified amino acid are identical to amino acid 41-211 of the wild-type tumor necrosis factor receptor-1 polypeptide shown in SEQ ID NO: 1; Selected from L68V/S92I/H95F/R97P/H98A, L68V/S92M/H95F/R97P/H98A, L68V/S92H/H95F/R97P/H98A, L68V/S92I/H95F/R97P/H98A/K161Q, L68V/S92I/H95F/ R97P/H98A/K161N, L68V/S92M/H95F/R97P/H98A/K161Q, L68V/S92M/H95F/R97P/H98A/K161N, L68V/S92H/H95F/R97P/H98A/K161Q, L68V/S92H/H95F/R97 Amino acid residue modification of H98A/K161N, L68V/S92I/H95F/R97P/H98G/K161Q and L68V/S92M/H95F/R97P/H98G/K161N, wherein the amino acid of the polypeptide except the modified amino acid is the same as shown in SEQ ID NO: 1 Amino acids 41-166 of the wild-type tumor necrosis factor receptor-1 polypeptide are identical; and Selected from L68V/S92I/H95F/R97P/H98A, L68V/S92M/H95F/R97P/H98A, L68V/S92H/H95F/R97P/H98A, L68V/S92I/H95F/R97P/H98G and L68V/S92M/H95F/R97P/ Amino acid residue modification of H98G, wherein the amino acid of the polypeptide except the modified amino acid is the same as amino acid 41-145 of the wild-type tumor necrosis factor receptor-1 polypeptide shown in SEQ ID NO: 1, and the polypeptide further comprises a sugar group selected from Chemical modifications of ylation, acylation, methylation, phosphorylation, hasylation, sulfation, prenylation, oxidation, guanidinylation, trinitrobenzoylation, nitration, and PEGylation. 5. The tumor necrosis factor receptor-1 polypeptide of modification according to claim 4, it is selected from SEQIDNO:44, SEQIDNO:49, SEQIDNO:61, SEQIDNO:62, SEQIDNO:64-72, SEQIDNO:75, SEQIDNO :78 and the amino acid sequences of SEQ ID NO:86-98. 6. The modified tumor necrosis factor receptor-1 polypeptide according to claim 4 or 5, wherein the acylation is amidation. 7. The modified tumor necrosis factor receptor-1 polypeptide of claim 4, wherein the acylation is carbamylation. 8. A gene encoding the modified tumor necrosis factor receptor-1 polypeptide of claim 1 or 2. 9. The gene according to claim 8, which is constructed based on the nucleotide sequence of SEQ ID NO: 5 with codon modifications carried out so that the gene can be expressed in Escherichia coli. 10. A vector carrying the gene of claim 8. 11. A cell transformed with the vector of claim 10. 12. The cell of claim 11, wherein the cell is E. coli. 13. A pharmaceutical formulation comprising the modified tumor necrosis factor receptor-1 polypeptide of claim 1 or 2. 14. A pharmaceutical composition for preventing or treating a TNF-mediated disease selected from the group consisting of adult respiratory distress syndrome, anorexia, cancer, chronic fatigue syndrome, graft-versus-host rejection, hyperalgesia, inflammatory Bowel disease, neuroinflammatory disease, ischemia-reperfusion injury, brain injury, diabetes, multiple sclerosis, eye disease, pain, pancreatitis, pulmonary fibrosis, rheumatoid arthritis, osteoarthritis, juvenile rheumatoid arthritis seronegative polyarthritis, ankylosing spondylitis, Reiter syndrome and reactive arthritis, psoriatic arthritis, enteropathic arthritis, polymyositis, dermatomyositis, scleroderma, systemic Sclerosis, vasculitis, cerebral vasculitis, Sjogren's syndrome, rheumatoid fever, polychondritis and polymyalgia, rheumatoid arteritis and giant cell arteritis, septic shock, side effects from radiotherapy, systemic Lupus erythematosus, temporomandibular joint dysfunction, thyroiditis and tissue transplantation, the pharmaceutical composition comprising the modified tumor necrosis factor receptor-1 polypeptide according to claim 1 or 2. 15. A pharmaceutical composition for preventing or treating a TNF-mediated disease selected from the group consisting of adult respiratory distress syndrome, anorexia, cancer, chronic fatigue syndrome, graft-versus-host rejection, hyperalgesia, inflammatory Bowel disease, neuroinflammatory disease, ischemia-reperfusion injury, brain injury, diabetes, multiple sclerosis, eye disease, pain, pancreatitis, pulmonary fibrosis, rheumatoid arthritis, osteoarthritis, juvenile rheumatoid arthritis seronegative polyarthritis, ankylosing spondylitis, Reiter syndrome and reactive arthritis, psoriatic arthritis, enteropathic arthritis, polymyositis, dermatomyositis, scleroderma, systemic Sclerosis, vasculitis, cerebral vasculitis, Sjogren's syndrome, rheumatoid fever, polychondritis and polymyalgia, rheumatoid arteritis and giant cell arteritis, septic shock, side effects from radiotherapy, systemic Lupus erythematosus, temporomandibular joint dysfunction, thyroiditis and tissue transplantation, the pharmaceutical composition comprises the gene of claim 8 or the carrier carrying the gene. 16. A method for producing the modified tumor necrosis factor receptor-1 polypeptide according to claim 1 or 2, which comprises using the gene according to claim 8 or a vector carrying the gene. 17. The modified tumor necrosis factor receptor-1 polypeptide according to claim 1, wherein the amino acid modification of the modified tumor necrosis factor receptor-1 polypeptide is L68V/S92M/H95F/R97P/H98G/K161N, wherein the polypeptide Other amino acids except the modified amino acid are the same as amino acid 41-211 of the wild-type tumor necrosis factor receptor-1 polypeptide represented by SEQ ID NO:1.